Combination immune therapy and cytokine control therapy for cancer treatment

ABSTRACT

Compositions disclosed herein, and methods of use thereof included those for inhibiting or reducing the incidence of cytokine release syndrome or cytokine storm in a subject undergoing CAR T-cell therapy, wherein the subjects are administered compositions including apoptotic cells or apoptotic cell supernatants. In certain instances compositions and methods of use thereof disclosed herein do not reduce the efficacy of the CAR T-cell cancer therapy. Disclosed herein are also compositions and methods of use thereof for decreasing or inhibiting cytokine production in a subject experiencing cytokine release syndrome or cytokine storm including administration of a composition including apoptotic cells or an apoptotic cell supernatant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase Application of PCT InternationalApplication Number PCT/IL2016/050194, International filing date Feb. 18,2016, which claims the benefit of U.S. Provisional Application Ser. No.62/117,752 filed Feb. 18, 2015, U.S. Provisional Application Ser. No.62/127,218 filed Mar. 2, 2015, U.S. Provisional Application Ser. No.62/148,227 filed Apr. 16, 2015, and U.S. Provisional Application Ser.No. 62/159,365 filed May 11, 2015, All of these applications are herebyincorporated by reference in their entirety herein.

FIELD OF INTEREST

Disclosed herein are compositions and methods thereof for inhibiting orreducing the incidence of cytokine release syndrome (CRS) or a cytokinestorm in a subject undergoing CAR T-cell cancer therapy. Further,disclosed herein are compositions and methods thereof for decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or a cytokine storm. Methods disclosed herein includethose comprising administration of a composition comprising apoptoticcells or an apoptotic cell supernatant in combination with a CAR T-celltherapy.

BACKGROUND

While standard treatments for cancer are surgery, chemotherapy, andradiation therapy, improved methods, such as targeted immunologicaltherapies, are currently being developed and tested. One promisingtechnique uses adoptive cell transfer (ACT), in which immune cells aremodified to recognize and attack their tumors. One example of ACT iswhen a patient's own cytotoxic T-cells, or a donor's, are engineered toexpress a chimeric antigen receptor (CAR T-cells) targeted to a tumorspecific antigen expressed on the surface of the tumor cells. These CART-cells are then cytotoxic only to cells expressing the tumor specificantigen. Clinical trials have shown that CAR T-cell therapy has greatpotential in controlling advanced acute lymphoblastic leukemia (ALL) andlymphoma, among others.

However, some patients given CAR T-cell therapy and other immunetherapies experience a dangerous and sometimes life-threatening sideeffect called cytokine release syndrome (CRS), in which the infused,activated T-cells produce a systemic inflammatory response in whichthere is a rapid and massive release of cytokines into the bloodstream,leading to dangerously low blood pressure, high fever and shivering.

In severe cases of CRS, patients experience a cytokine storm (a.k.a.cytokine cascade or hypercytokinemia), in which there is a positivefeedback loop between cytokines and white blood cells with highlyelevated levels of cytokines. This can lead to potentiallylife-threatening complications including cardiac dysfunction, adultrespiratory distress syndrome, neurologic toxicity, renal and/or hepaticfailure, pulmonary edema and disseminated intravascular coagulation.

For example, six patients in a recent phase I trial who wereadministered the monoclonal antibody TGN1412, which binds to the CD28receptor on T-cells, exhibited severe cases of cytokine storm andmulti-organ failure. This happened despite the fact that the TGN1412dose was 500-times lower than that found to be safe in animals (St.Clair E W: The calm after the cytokine storm: Lessons from the TGN1412trial. J Clin Invest 118: 1344-1347, 2008).

To date, corticosteroids, biological therapies such as anti-IL6therapies and anti-inflammatory drugs are being evaluated to controlcytokine release syndrome in patients administered CAR T-cell therapy.However, steroids may affect CAR T-cells' activity and/or proliferationand put the patients in danger of sepsis and opportunistic infections.Anti-inflammatory drugs may not be effective in controlling cytokinerelease syndromes or cytokine storms, because the cytokine stormincludes a very large number of cytokines while there is limited abilityto infuse patients with anti-inflammatory drugs. Novel strategies areneeded to control cytokine release syndromes, and especially cytokinestorms, in order to realize the potential of CAR T-cell therapy.

Cytokine storms are also a problem after other infectious andnon-infectious stimuli. In a cytokine storm, numerous proinflammatorycytokines, such as interleukin-1 (IL-1), IL-6, g-interferon (g-IFN), andtumor necrosis factor-α (TNFα), are released, resulting in hypotension,hemorrhage, and, ultimately, multiorgan failure. The relatively highdeath rate in young people, with presumably healthy immune systems, inthe 1918 H1N1 influenza pandemic and the more recent bird flu H5N1infection are attributed to cytokine storms. This syndrome has been alsoknown to occur in advanced or terminal cases of severe acute respiratorysyndrome (SARS), Epstein-Barr virus-associated hemophagocyticlymphohistiocytosis, gram-negative sepsis, malaria and numerous otherinfectious diseases, including Ebola infection.

Cytokine storm may also stem from non-infectious causes, such as acutepancreatitis, severe burns or trauma, or acute respiratory distresssyndrome. Novel strategies are therefore needed to control cytokinerelease syndrome, and especially cytokine storms.

SUMMARY

In one aspect, disclosed herein is a composition comprising chimericantigen receptor-expressing T-cells (CAR T-cells), and either apoptoticcells or an apoptotic cell supernatant, and a pharmaceuticallyacceptable excipient. In a related aspect, apoptotic cells compriseapoptotic cells in an early apoptotic state. In another related aspect,the apoptotic cells comprise pooled third party donor cells. In arelated aspect, an apoptotic cell supernatant is obtained by a methodcomprising the steps of (a) providing apoptotic cells, (b) culturing thecells of step (a), and (c) separating the supernatant from the cells. Ina related aspect, the apoptotic cell supernatant is an apoptoticcell-white blood cell supernatant and said obtaining further comprisesthe steps of: (d) providing white blood cells, (e) optionally, washingthe apoptotic cells and the white blood cells, (f) co-culturing theapoptotic cells and the white blood cells, wherein steps (d)-(f) are inplace of step (b). In another related aspect, the provided white bloodcells are selected from the group consisting of phagocytes, macrophages,dendritic cells, monocytes, B cells, T cells, and NK cells.

In a related aspect, the CAR T-cells and the either apoptotic cells orapoptotic cell supernatant are comprised in separate compositions.

In a related aspect, the composition further comprises an additionalagent selected from the group comprising a CTLA-4 blocking agent, analpha-1 anti-trypsin or fragment thereof or analog thereof, atellurium-based compound, or an immune modulating agent, or anycombination thereof. In another related aspect, the additional agent orany combination thereof is comprised in a composition with the CART-cells, or with either the apoptotic cells or the apoptotic cellsupernatant, or in another related aspect is comprised in a separatecomposition.

In one aspect, disclosed herein is a method of inhibiting or reducingthe incidence of a cytokine release syndrome (CRS) or a cytokine stormin a subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) cancer therapy, the method comprising the step of administeringa composition comprising apoptotic cells or an apoptotic cellsupernatant to said subject, wherein said administration inhibits orreduces the incidence of the CRS or cytokine storm in the subject. In arelated aspect, the apoptotic cells comprise apoptotic cells in anearly-apoptotic state. In another related aspect, the apoptotic cellsare autologous to the subject or are pooled third-party donor cells. Inanother related aspect, the administration of said compositioncomprising said apoptotic cells or said apoptotic cell supernatantoccurs prior to, concurrent with, or following the CAR T-cell therapy.In another related aspect, the apoptotic cell supernatant is obtained bya method comprising the steps of: (a) providing apoptotic cells, (b)culturing the cells of step (a), and (c) separating the supernatant fromthe cells. In another related aspect, said apoptotic cell supernatant isan apoptotic cell-white blood cell supernatant and said method furthercomprises the steps of: (d) providing white blood cells, (e) optionally,washing the apoptotic cells and the white blood cells, (f) co-culturingthe apoptotic cells and the white blood cells, wherein steps (d)-(f) arein place of step (b). In another related aspect, the provided whiteblood cells are selected from the group consisting of phagocytes,macrophages, dendritic cells, monocytes, B cells, T cells, and NK cells.

In another related aspect, the method further comprises administering anadditional agent selected from the group comprising a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment thereof or analogue thereof,a tellurium-based compound, or an immune modulating agent, or anycombination thereof. In another related aspect, the administration ofsaid additional agent occurs prior to, concurrent with, or following theCAR T-cell therapy. In another related aspect, the level ofpro-inflammatory cytokines are reduced in the subject compared with asubject undergoing CAR T-cell cancer therapy and not administered saidapoptotic cells or said apoptotic cell supernatant.

In one aspect, disclosed herein is a method of decreasing or inhibitingcytokine production in a subject experiencing cytokine release syndromeor cytokine storm or vulnerable to cytokine release syndrome or cytokinestorm, comprising the step of administering a composition comprisingapoptotic cells or an apoptotic cell supernatant to said subject,wherein said administering decreases or inhibits cytokine production insaid subject. In a related aspect, the production of at least onepro-inflammatory cytokine is decreased or inhibited in said subjectcompared with a subject experiencing cytokine release syndrome orcytokine storm or vulnerable to cytokine release syndrome or cytokinestorm and not administered apoptotic cells or an apoptotic cellsupernatant. In another related aspect, the subject is undergoing CART-cell cancer therapy and said method does not reduce the efficacy ofsaid CAR T-cell cancer therapy.

In a related aspect, disclosed herein the cause of said cytokine releasesyndrome or cytokine storm comprises an infectious stimuli, condition,or syndrome. In another related aspect, the infectious stimuli,condition, or syndrome comprises influenza, bird flu, severe acuterespiratory syndrome (SARS), Epstein-Barr virus-associatedhemophagocytic lymphohistiocytosis (HLH), sepsis, gram-negative sepsis,malaria, an Ebola virus, a variola virus, a systemic Gram-negativebacterial infection, or Jarisch-Herxheimer syndrome.

In a related aspect, the cause of said cytokine release syndrome orcytokine storm comprises a non-infectious stimuli, condition, orsyndrome. In another related aspect, the non-infectious stimuli,condition, or syndrome comprises is hemophagocytic lymphohistiocytosis(HLH), sporadic HLH, macrophage activation syndrome (MAS), chronicarthritis, systemic Juvenile Idiopathic Arthritis (sJIA), Still'sDisease, a Cryopyrin-associated Periodic Syndrome (CAPS), Familial ColdAuto-inflammatory Syndrome (FCAS), Familial Cold Urticaria (FCU),Muckle-Well Syndrome (MWS), Chronic Infantile Neurological Cutaneous andArticular (CINCA) Syndrome, a cryopyrinopathy comprising inherited or denovo gain of function mutations in the NLRP3 gene, a hereditaryauto-inflammatory disorder, acute pancreatitis, a severe burns, atrauma, an acute respiratory distress syndrome, an immunotherapy, amonoclonal antibody therapy, secondary to drug use, is secondary toinhalation of toxins, a lipopolysaccharide (LPS), a Gram-positivetoxins, fungal toxins, glycosylphosphatidylinositol (GPI), or modulationof RIG-1 gene expression.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter disclosed herein is particularly pointed out anddistinctly claimed in the concluding portion of the specification. Thecompositions and methods disclosed herein, however, both as toorganization and method of operation, together with objects, features,and advantages thereof, may best be understood by reference to thefollowing detailed description when read with the accompanying drawings.

FIGS. 1A-1B. Schematic showing standard CAR T-cell therapy (FIG. 1A) andembodiments of a method of safe and efficacious CAR T-cell cancertherapy in a patient using patients' own cells (autologous) (FIG. 1B) toproduce apoptotic cells or an apoptotic cell supernatant.

FIG. 2. Schematic showing embodiments of a method of safe andefficacious CAR T-cell cancer therapy in a patient, using donor cells toproduce apoptotic cells or an apoptotic supernatant.

FIG. 3. Verification of Transduction of T-cells showing the flowcytometry results of anti-CD124 analysis of transduced T4⁺ CAR-T cells.

FIG. 4. T4⁺CAR T-Cells reduced proliferation of SKOV3-luc ovarianadenocarcinoma cells. The results of the cytotoxicity assay, wherein amonolayer of SKOV3-luc cells were cultured either by non-transduced Tcells or by T4+ CAR-T cells, are presented in a bar graph.

FIG. 5. Apoptotic Cells do not abrogate T4⁺ CAR-T cells anti-tumoractivity. Results are based on a cytotoxicity assay, wherein a monolayerof SKOV3-luc cells were cultured either with non-transduced T cells orwith T4⁺CAR-T cells in the presence of a vehicle (Hartmann solution), orapoptotic cells (Apocell), or a supernatant of apoptotic cells (ApoSup),or supernatant of co-culture of apoptotic cells andmonocytes/macrophages (ApoMon Sup).

FIG. 6. Il-6, secreted at high levels during cytotoxicity, isdown-regulated by apoptotic cells. The results shown here demonstratethe effect of co-culture of SKOV3-luc and human monocytes/macrophageswere exposed to apoptotic cells (ApoCell), or ApoCell supernatant(ApoSup), or apoptotic cells and monocyte/macrophage co-culture (ApoMonSup).

FIG. 7. Effect of Apoptotic Cells or Apoptotic Cell Supernatant or aco-culture of Apoptotic cells and Monocytes following LPS exposureduring CAR-T cell therapy. Extremely high secretion of IL-6 wasdocumented when lipopolysaccharides (LPS) were added to the cytotoxicassay. Results show that exposure to Apoptotic cells (Apocell), orsupernatant of apoptotic cells (ApoSup) or supernatant of co-culture ofapoptotic cells and monocytes/macrophages (ApoMon Sup), down regulatedIL-6, wherein IL-6 was reduced to acceptable levels.

It will be appreciated that for simplicity and clarity of illustration,elements shown in the figures have not necessarily been drawn to scale.For example, the dimensions of some of the elements may be exaggeratedrelative to other elements for clarity. Further, where consideredappropriate, reference numerals may be repeated among the figures toindicate corresponding or analogous elements.

DETAILED DESCRIPTION

This application claims the benefit of U.S. Patent ProvisionalApplications No. 62/117,752, filed Feb. 18, 2015; 62/127,218, filed Mar.2, 2015, 62/148,227, filed Apr. 16, 2015; and 62/159,365, filed May 11,2015. These applications are hereby incorporated by reference in theirentirety herein.

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the methodsdisclosed herein. However, it will be understood by those skilled in theart that these methods may be practiced without these specific details.In other instances, well-known methods, procedures, and components havenot been described in detail so as not to obscure the methods disclosedherein.

Genetic modification of immune cells is well known as a strategy forimmune-cell therapies against cancer. These immune-cell therapies arebased on the manipulation and administration of autologous or allogeneicimmune cells to a subject in need. Immune-cell based therapies includenatural killer cells therapies, dendrite cell therapies, and T-cellimmunotherapies including those utilizing naïve T-cells, effectorT-cells also known as T-helper cells, cytotoxic T-cells, and regulatoryT-cells (Tregs).

In one embodiment, disclosed herein are compositions comprisinggenetically modified immune cells In another embodiment, the geneticallymodified immune cell is a T-cell. In another embodiment, a T-cell is anaïve T-cell. In another embodiment, a T-cell is a naïve CD4⁺ T-cell. Inanother embodiment, a T-cell is a naïve T-cell. In another embodiment, aT-cell is a naïve CD8⁺ T-cell. In another embodiment, the geneticallymodified immune cell is a natural killer (NK) cell. In anotherembodiment, the genetically modified immune cell is a dendritic cell. Instill another embodiment, the genetically modified T-cell is a cytotoxicT lymphocyte (CTL cell). In another embodiment, the genetically modifiedT-cell is a regulatory T-cell (Treg). In another embodiment, thegenetically modified T-cell is a chimeric antigen receptor (CAR) T-cell.In another embodiment, the genetically modified T-cell is a geneticallymodified T-cell receptor (TCR) cell.

In one embodiment, disclosed herein are compositions comprisinggenetically modified immune cells and apoptotic cells. In anotherembodiment, disclosed herein are compositions comprising geneticallymodified immune cells and supernatants from apoptotic cells. In anotherembodiment, the genetically modified immune cell is a T-cell. In anotherembodiment, the genetically modified immune cell is a natural killer(NK) cell. In still another embodiment, the genetically modified immunecell is a cytotoxic T lymphocyte (CTL cell). In another embodiment, thegenetically modified immune cell is a regulatory T lymphocyte (Tregcell).

In one embodiment, disclosed herein are compositions comprisinggenetically modified T-cells and apoptotic cells. In another embodiment,disclosed herein are compositions comprising genetically modifiedT-cells and supernatants of apoptotic cells. In another embodiment, thegenetically modified T-cell is a chimeric antigen receptor (CAR) T-cell.In another embodiment, the genetically modified T-cell is a geneticallymodified T-cell receptor (TCR) cell.

In one embodiment, disclosed herein are compositions comprising CART-cells and apoptotic cells. In another embodiment, disclosed herein arecompositions comprising genetically modified T-cell receptor cells(TCRs) and apoptotic cells. In another embodiment, disclosed herein arecompositions comprising CAR T-cells and supernatants from apoptoticcells. In another embodiment, disclosed herein are compositionscomprising genetically modified T-cell receptor cells (TCRs) andsupernatant of apoptotic cells.

In certain embodiments, genetically modified immune cells and apoptoticcells or apoptotic cell supernatants are comprised within a singlecomposition. In other embodiments, genetically modified immune cells andapoptotic cells or apoptotic cell supernatants are comprised in separatecompositions.

In one embodiment, disclosed herein are compositions comprisinggenetically modified immune cells and an additional agent selected fromthe group comprising a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment thereof or analogue thereof, a tellurium-based compound, or animmune modulating agent, or any combination thereof. In anotherembodiment, disclosed herein are compositions comprising geneticallymodified immune cells, apoptotic cells, and an additional agent selectedfrom the group comprising a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment thereof or analogue thereof, a tellurium-basedcompound, or an immune modulating agent, or any combination thereof. Inanother embodiment, disclosed herein are compositions comprisinggenetically modified immune cells and an additional agent selected fromthe group comprising a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment thereof or analogue thereof, a tellurium-based compound, or animmune modulating agent, or any combination thereof. In anotherembodiment, disclosed herein are compositions comprising geneticallymodified immune cells, supernatants from apoptotic cells, and anadditional agent selected from the group comprising a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment thereof or analogue thereof,a tellurium-based compound, or an immune modulating agent, or anycombination thereof. In another embodiment, the genetically modifiedimmune cell is a T-cell. In another embodiment, the genetically modifiedimmune cell is a natural killer (NK) cell. In still another embodiment,the genetically modified immune cell is a cytotoxic T lymphocyte (CTLcell).

In one embodiment, disclosed herein are compositions comprisinggenetically modified T-cells and an additional agent selected from thegroup comprising a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment thereof or analogue thereof, a tellurium-based compound, or animmune modulating agent, or any combination thereof. In anotherembodiment, disclosed herein are compositions comprising geneticallymodified T-cells, apoptotic cells, and an additional agent selected fromthe group comprising a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment thereof or analogue thereof, a tellurium-based compound, or animmune modulating agent, or any combination thereof. In anotherembodiment, disclosed herein are compositions comprising geneticallymodified T-cells and an additional agent selected from the groupcomprising a CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragmentthereof or analogue thereof, a tellurium-based compound, or an immunemodulating agent, or any combination thereof. In another embodiment,disclosed herein are compositions comprising genetically modifiedT-cells, supernatants of apoptotic cells, and an additional agentselected from the group comprising a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment thereof or analogue thereof, a tellurium-basedcompound, or an immune modulating agent, or any combination thereof. Inanother embodiment, the genetically modified T-cell is a chimericantigen receptor (CAR) T-cell. In another embodiment, the geneticallymodified T-cell is a genetically modified T-cell receptor (TCR) cell.

In one embodiment, disclosed herein are compositions comprising CART-cells and an additional agent selected from the group comprising aCTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment thereof oranalogue thereof, a tellurium-based compound, or an immune modulatingagent, or any combination thereof. In another embodiment, disclosedherein are compositions comprising CAR T-cells, apoptotic cells, and anadditional agent selected from the group comprising a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment thereof or analogue thereof,a tellurium-based compound, or an immune modulating agent, or anycombination thereof. In another embodiment, disclosed herein arecompositions comprising genetically modified T-cell receptors (TCRs) andan additional agent selected from the group comprising a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment thereof or analogue thereof,a tellurium-based compound, or an immune modulating agent, or anycombination thereof. In another embodiment, disclosed herein arecompositions comprising genetically modified T-cell receptors (TCRs),apoptotic cells, and an additional agent selected from the groupcomprising a CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragmentthereof or analogue thereof, a tellurium-based compound, or an immunemodulating agent, or any combination thereof. In another embodiment,disclosed herein are compositions comprising genetically modified T-cellreceptors (TCRs), apoptotic cell supernatants, and an additional agentselected from the group comprising a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment thereof or analogue thereof, a tellurium-basedcompound, or an immune modulating agent, or any combination thereof.

In one embodiment, administration of a composition comprising apoptoticcells does not affect the efficacy of CAR T-cells to treat, prevent,inhibit, reduce the incidence of, ameliorating, or alleviating a canceror a tumor. In another embodiment, administration of a compositioncomprising apoptotic cells does not reduce the efficacy of the CART-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 5%. In another embodiment, administration of a compositioncomprising apoptotic cells does not reduce the efficacy of the CART-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 10%. In another embodiment, administration of a compositioncomprising apoptotic cells does not reduce the efficacy of the CART-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 15%. In another embodiment, administration of a compositioncomprising apoptotic cells does not reduce the efficacy of the CART-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 20%.

In another embodiment, administration of a composition comprising anapoptotic cell supernatant does not reduce the efficacy of the CART-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 5%. In another embodiment, administration of a compositioncomprising an apoptotic cell supernatant does not reduce the efficacy ofthe CAR T-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 10%. In another embodiment, administration of a compositioncomprising an apoptotic cell supernatant does not reduce the efficacy ofthe CAR T-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 15%. In another embodiment, administration of a compositioncomprising an apoptotic cell supernatant does not reduce the efficacy ofthe CAR T-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 20%. In another embodiment, administration of a compositioncomprising the apoptotic cell supernatant does not affect the efficacyof the CAR T-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor. In anotherembodiment, administration of a composition comprising the apoptoticcell supernatant does not reduce the efficacy of the CAR T-cells totreat, prevent, inhibit, reduce the incidence of, ameliorating, oralleviating said cancer or said tumor.

In another embodiment, disclosed herein are methods of inhibiting orreducing the incidence of cytokine release syndrome (CRS) or cytokinestorm in a subject undergoing CAR T-cell cancer therapy. In anotherembodiment, methods disclosed herein decrease or prevent cytokineproduction in a subject undergoing CAR T-cell cancer therapy therebyinhibiting or reducing the incidence of cytokine release syndrome (CRS)or cytokine storm in a subject. In another embodiment, the methodsdisclosed herein of inhibiting or reducing the incidence of cytokinerelease syndrome (CRS) or cytokine storm in a subject undergoing CART-cell cancer therapy comprise the step of administering a compositioncomprising apoptotic cells to the subject undergoing the cancer therapy.In yet another embodiment, methods disclosed herein for decreasing orinhibiting cytokine production in a subject undergoing CAR T-cell cancertherapy comprise the step of administering a composition comprisingapoptotic cells to the subject undergoing the cancer therapy. In anotherembodiment, administration of a composition comprising apoptotic cellsdoes not affect the efficacy of the CAR T-cell therapy. In anotherembodiment, administration of a composition comprising apoptotic cellsor an apoptotic supernatant does not reduce the efficacy of the CART-cell therapy. In another embodiment, administration of a compositioncomprising apoptotic cells or an apoptotic cell supernatant does notreduce the efficacy of the CAR T-cells therapy by more than about 5%. Inanother embodiment, administration of a composition comprising apoptoticcells or an apoptotic cell supernatant does not reduce the efficacy ofthe CAR T-cells therapy by more than about 10%. In another embodiment,administration of a composition comprising apoptotic cells or anapoptotic cell supernatant does not reduce the efficacy of the CART-cells therapy by more than about 15%. In another embodiment,administration of a composition comprising apoptotic cells or anapoptotic cell supernatant does not reduce the efficacy of the CART-cells therapy by more than about 20%.

In another embodiment, disclosed herein are methods of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm comprising the step of administering anapoptotic cell supernatant, as disclosed herein, or a compositioncomprising said apoptotic cell supernatant. In another embodiment, anapoptotic cell supernatant comprises an apoptotic cell-phagocytesupernatant.

In still another embodiment, methods disclosed herein for decreasing orinhibiting cytokine production in a subject undergoing CAR T-cell cancertherapy comprise the step of administering a composition comprising anapoptotic cell supernatant to the subject undergoing the cancer therapy.In another embodiment, administration of a composition comprising anapoptotic cell supernatant does not affect the efficacy of the CART-cell therapy. In another embodiment, administration of a compositioncomprising an apoptotic cell supernatant does not reduce the efficacy ofthe CAR T-cell therapy.

In one embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing chimeric antigen receptor-expressing T-cell (CAR T-cell)cancer therapy comprises the step of administering a compositioncomprising apoptotic cells or an apoptotic supernatant to said subject.In another embodiment, a method of inhibiting or reducing the incidenceof a cytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing chimeric antigen receptor-expressing T-cell (CAR T-cell)cancer therapy decreases or inhibits production of at least onepro-inflammatory cytokine in the subject.

Chimeric Antigen Receptor-Expressing T-Cells (CAR T-Cells)

In one embodiment, chimeric antigen receptors (CARs) are a type ofantigen-targeted receptor composed of intracellular T-cell signalingdomains fused to extracellular tumor-binding moieties, most commonlysingle-chain variable fragments (scFvs) from monoclonal antibodies. CARsdirectly recognize cell surface antigens, independent of MHC-mediatedpresentation, permitting the use of a single receptor construct specificfor any given antigen in all patients. Initial CARs fusedantigen-recognition domains to the CD3ζ activation chain of the T-cellreceptor (TCR) complex. While these first generation CARs induced T-celleffector function in vitro, they were largely limited by poor antitumorefficacy in vivo. Subsequent CAR iterations have included secondarycostimulatory signals in tandem with CD3ζ, including intracellulardomains from CD28 or a variety of TNF receptor family molecules such as4-1BB (CD137) and OX40 (CD134). Further, third generation receptorsinclude two costimulatory signals in addition to CD3ζ, most commonlyfrom CD28 and 4-1BB. Second and third generation CARs dramaticallyimproved antitumor efficacy, in some cases inducing complete remissionsin patients with advanced cancer.

In one embodiment, a CAR T-cell is an immunoresponsive cell comprisingan antigen receptor, which is activated when its receptor binds to itsantigen.

In one embodiment, the CAR T-cells used in the compositions and methodsas disclosed herein are first generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are second generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are third generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are fourth generation CAR T-cells. In one embodiment,each generation of CAR T-cells is more potent than the CAR T-cells ofearlier generations.

In one embodiment, first-generation CARs have one signaling domain,typically the cytoplasmic signaling domain of the CD3 TCRζ chain.

In another embodiment, the CAR T-cells as disclosed herein are secondgeneration CAR T-cells. In another embodiment, CAR T-cells as disclosedherein comprise a tripartite chimeric receptor (TPCR). In oneembodiment, CAR T-cells as disclosed herein, comprise one or moresignaling moieties that activate naïve T-cells in a co-stimulationindependent manner. In another embodiment, the CAR T-cells furtherencode one or more members of the tumor necrosis factor receptor family,which in one embodiment, is CD27, 4-1BB (CD137), or OX40 (CD134), or acombination thereof.

Third-generation CAR T-cells attempt to harness the signaling potentialof 2 costimulatory domains: in one embodiment, the CD28 domain followedby either the 4-1BB or OX-40 signaling domains. In another embodiment,the CAR T-cells used in the compositions and methods as disclosed hereinfurther encode a co-stimulatory signaling domain, which in oneembodiment is CD28. In another embodiment, the signaling domain is theCD3ζ-chain, CD97, GDI 1a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB,CD28 signaling domain, or combinations thereof.

In one embodiment, telomere length and replicative capacity correlatewith the engraftment efficiency and antitumor efficacy of adoptivelytransferred T-cell lines. In one embodiment, CD28 stimulation maintainstelomere length in T-cells.

In one embodiment, CAR-modified T-cell potency may be further enhancedthrough the introduction of additional genes, including those encodingproliferative cytokines (ie, IL-12) or costimulatory ligands (ie,4-1BBL), thus producing “armored” fourth-generation CAR-modifiedT-cells. In one embodiment, “armored CAR T-cells,” are CAR T-cells whichare protected from the inhibitory tumor microenvironment. In anotherembodiment, the “armored” CAR technology incorporates the localsecretion of soluble signaling proteins to amplify the immune responsewithin the tumor microenvironment with the goal of minimizing systemicside effects. In one embodiment, the signaling protein signal is IL-12,which can stimulate T-cell activation and recruitment. In oneembodiment, “armored” CAR technology is especially useful in solid tumorindications, in which microenvironment and potent immunosuppressivemechanisms have the potential to make the establishment of a robustanti-tumor response more challenging.

In one embodiment, CAR T-cells are genetically modified to encodemolecules involved in the prevention of apoptosis, the remodeling of thetumor microenvironment, induction of homeostatic proliferation, andchemokine receptors that promote directed T-cell homing.

In another embodiment, CAR T-cell therapy used in the compositions andmethods as disclosed herein is enhanced using the expression of cytokinetransgenes, combination therapy with small molecule inhibitors, ormonoclonal antibodies. In another embodiment, other strategies aimed atimproving CAR T-cell therapy including using dual CARs and chemokinereceptors to more specifically target tumor cells are to be consideredpart of the CAR T-cells and CAR T-cell therapy as disclosed herein.

In one embodiment, the CAR T-cells of the compositions and methods asdisclosed herein comprise a second binding domain that can lead toeither an inhibitory or amplifying signal, in order to increasespecificity of CAR T-cells for cancer cells versus normal cells. Forexample, a CAR T-cell can be engineered such that it would be triggeredin the presence of one target protein, but if a second protein ispresent it would be inhibited. Alternatively, it could also beengineered such that two target proteins would be required for maximalactivation. These approaches may increase the specificity of the CAR fortumor relative to normal tissue.

In one embodiment, the CAR T-cells used in the compositions and methodsas disclosed herein encode antibody-based external receptor structuresand cytosolic domains that encode signal transduction modules composedof the immunoreceptor tyrosine-based activation motif.

In one embodiment, the CAR T-cell further encodes a single-chainvariable fragment (scFv) that binds a polypeptide that hasimmunosuppressive activity. In another embodiment, the polypeptide thathas immunosuppressive activity is CD47, PD-1, CTLA-4, or a combinationthereof.

In one embodiment, the CAR T-cell further encodes a single-chainvariable fragment (scFv) that binds a polypeptide that hasimmunostimulatory activity. In another embodiment, the polypeptide thathas immunostimulatory activity is CD28, OX-40, 4-1 BB or a combinationthereof. In another embodiment, the CAR T-cell further encodes a CD40ligand (CD40L), which, in one embodiment, enhances the immunostimulatoryactivity of the antigen.

In one embodiment, the immune cells are cytotoxic. In anotherembodiment, cytotoxic cells for genetic modification can be obtainedfrom bone marrow of the subject or a donor. In other cases, the cellsare obtained from a stein cell. For example, cytotoxic cells can bederived from human pluripotent stem cells such as human embryonic stemcells or human induced pluripotent T-cells. In the case of inducedpluripotent stem cells (IPSCs), such pluripotent T-cells can be obtainedusing a somatic cell from the subject to which genetically modifiedcytotoxic cells will be provided. In one embodiment, immune cells may beobtained from a subject or donor by harvesting cells by venipuncture, byapheresis methods, by white cell mobilization followed by apheresis orvenipuncture, or by bone marrow aspiration.

In one embodiment, a method as disclosed herein comprises obtainingimmune cells from a subject, and genetically modifying the immune cellsto express a chimeric antigen receptor. In another embodiment, a methodas disclosed herein comprises obtaining immune cells from a subject,genetically modifying the immune cells to express a chimeric antigenreceptor and combining with apoptotic cell population resulting inreduced cytokine production in a subject but substantially unaffectedcytotoxicity relative to immune cells expressing a CAR not administeredwith an apoptotic cell population (FIGS. 1A-1B and 2). In anotherembodiment, a method as disclosed herein comprises obtaining immunecells from a subject, genetically modifying the immune cells to expressa chimeric antigen receptor and combining with an apoptotic cellsupernatant or a composition comprising the supernatant, resulting inreduced cytokine production in a subject but substantially unaffectedcytotoxicity relative to immune cells expressing a CAR not administeredwith an apoptotic cell supernatant. In another embodiment,administration of an apoptotic cell population or a supernatant fromapoptotic cells does not reduce the efficacy of the immune cellsexpressing the chimeric antigen receptor.

Accordingly, one embodiment as disclosed herein relates to cytotoxicimmune cells (e.g., NK cells or T-cells) comprising chimeric antigenreceptors (CARs) whereby the cells retain their cytotoxic function. Inanother embodiment, the chimeric antigen receptor is exogenous to theT-cell. In another embodiment, the CAR is recombinantly expressed. Inanother embodiment, the CAR is expressed from a vector.

In one embodiment, the T-cell utilized to generate CAR T-cells is anaïve CD4⁺ T-cell. In another embodiment, the T-cell utilized togenerate CAR T-cells is a naïve CD8⁺ T-cell. In another embodiment, theT-cell utilized to generate CAR T-cells is an effector T-cell. Inanother embodiment, the T-cell utilized to generate CAR T-cells is aregulatory T-cell (Treg). In another embodiment, the T-cell utilized togenerate CAR T-cells is a cytotoxic T-cell.

Sources for genetically modified immune cells, for example T cells, havebeen described extensively in the literature, see for example Themelliet al. (2015) New Cell Sources for T Cell Engineering and AdoptiveImmunotherapy. Cell Stem Cell 16: 357-366; Han et al. (2013) Journal ofHematology & Oncology 6:47-53; Wilkie et al. (2010) J Bio Chem285(33):25538-25544; and van der Stegen et al. (2013) J. Immunol 191:4589-4598. CAR T-cells are available to order from a commercial sourcesuch as Creative Biolabs (NY USA), which provides custom constructionand production services for Chimeric Antigen Receptors (CAR) and alsoprovides premade CAR constructs stock, which can induce protectiveimmunity encode by recombinant adenovirus vaccine. Custom made CART-cells may also be obtained from Promab Biotechnologies (CA USA), whichcan provide specifically designed CAR T-cells.

Targeting Antigens

In one embodiment, the CAR binds to an epitope of an antigen via anantibody or an antibody fragment that is directed to the antigen. Inanother embodiment, the antibody is a monoclonal antibody. In anotherembodiment, the antibody is a polyclonal antibody. In anotherembodiment, the antibody fragment is a single-chain variable fragment(scFv).

In another embodiment, the CAR T-cells of the compositions as disclosedherein bind to a tumor associated antigen (TAA). In another embodiment,said tumor associated antigen is: Mucin 1, cell surface associated(MUC1) or polymorphic epithelial mucin (PEM), Arginine-rich, mutated inearly stage tumors (Armet), Heat Shock Protein 60 (HSP60), calnexin(CANX), methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2), fibroblast activationprotein (FAP), matrix metallopeptidase (MMP6), B Melanoma Antigen-1(BAGE-1), aberrant transcript of N-acetyl glucosaminyl transferase V(GnTV), Q5H943, Carcinoembryonic antigen (CEA), Pmel, Kallikrein-4,Mammaglobin-1, MART-1, GPR143-OA1, prostate specific antigen (PSA),TRP1, Tyrosinase, FGP-5, NEU proto-oncogene, Aft, MMP-2, prostatespecific membrane antigen (PSMA), Telomerase-associated protein-2,Prostatic acid phosphatase (PAP), Uroplakin II or Proteinase 3.

In another embodiment, the CAR binds to CD19 or CD20 to target B cellsin the case where one would like to destroy B cells as in leukemia. Inanother embodiment, the CAR binds to ROR1, CD22, or GD2. In anotherembodiment, the CAR binds to NY-ESO-1. In another embodiment, the CARbinds to MAGE family proteins. In another embodiment, the CAR binds tomesothelin. In another embodiment, the CAR binds to c-erbB2. In anotherembodiment, the CAR binds to mutational antigens that are tumorspecific, such as BRAFV600E mutations and BCR-ABL translocations. Inanother embodiment, the CAR binds to viral antigens which aretumor-specific, such as EBV in HD, HPV in cervical cancer, andpolyomavirus in Merkel cancer. In another embodiment, the CAR T-cellbinds to Her2/neu. In another embodiment, the CAR T-cell binds toEGFRvIII.

In one embodiment, the chimeric antigen receptor (CAR) T-cell binds theCD19 antigen. In another embodiment, the CAR binds the CD22 antigen. Inanother embodiment, the CAR binds to alpha folate receptor. In anotherembodiment, the CAR binds to CAIX. In another embodiment, the CAR bindsto CD20. In another embodiment, the CAR binds to CD23. In anotherembodiment, the CAR binds to CD24. In another embodiment, the CAR bindsto CD30. In another embodiment, the CAR binds to CD33. In anotherembodiment, the CAR binds to CD38. In another embodiment, the CAR bindsto CD44v6. In another embodiment, the CAR binds to CD44v7/8. In anotherembodiment, the CAR binds to CD123. In another embodiment, the CAR bindsto CD171. In another embodiment, the CAR binds to carcinoembryonicantigen (CEA). In another embodiment, the CAR binds to EGFRvIII. Inanother embodiment, the CAR binds to EGP-2. In another embodiment, theCAR binds to EGP-40. In another embodiment, the CAR binds to EphA2. Inanother embodiment, the CAR binds to Erb-B2. In another embodiment, theCAR binds to Erb-B 2, 3, 4. In another embodiment, the CAR binds toErb-B3/4. In another embodiment, the CAR binds to FBP. In anotherembodiment, the CAR binds to fetal acetylcholine receptor. In anotherembodiment, the CAR binds to G_(D2). In another embodiment, the CARbinds to G_(D3). In another embodiment, the CAR binds to HER2. Inanother embodiment, the CAR binds to HMW-MAA. In another embodiment, theCAR binds to IL-11Ralpha. In another embodiment, the CAR binds toIL-13Ralpha1. In another embodiment, the CAR binds to KDR. In anotherembodiment, the CAR binds to kappa-light chain. In another embodiment,the CAR binds to Lewis Y. In another embodiment, the CAR binds toL1-cell adhesion molecule. In another embodiment, the CAR binds toMAGE-A1. In another embodiment, the CAR binds to mesothelin. In anotherembodiment, the CAR binds to CMV infected cells. In another embodiment,the CAR binds to MUC1. In another embodiment, the CAR binds to MUC16. Inanother embodiment, the CAR binds to NKG2D ligands. In anotherembodiment, the CAR binds to NY-ESO-1 (amino acids 157-165). In anotherembodiment, the CAR binds to oncofetal antigen (h5T4). In anotherembodiment, the CAR binds to PSCA. In another embodiment, the CAR bindsto PSMA. In another embodiment, the CAR binds to ROR1. In anotherembodiment, the CAR binds to TAG-72. In another embodiment, the CARbinds to VEGF-R2 or other VEGF receptors. In another embodiment, the CARbinds to B7-H6. In another embodiment, the CAR binds to CA9. In anotherembodiment, the CAR binds to α_(v)β₆ integrin. In another embodiment,the CAR binds to 8H9. In another embodiment, the CAR binds to NCAM. Inanother embodiment, the CAR binds to fetal acetylcholine receptor.

In another embodiment, the chimeric antigen receptor (CAR) T-celltargets the CD19 antigen, and has a therapeutic effect on subjects withB-cell malignancies, ALL, Follicular lymphoma, CLL, and Lymphoma. Inanother embodiment, the CAR T-cell targets the CD22 antigen, and has atherapeutic effect on subjects with B-cell malignancies. In anotherembodiment, the CAR T-cell targets alpha folate receptor or folatereceptor alpha, and has a therapeutic effect on subjects with ovariancancer or epithelial cancer. In another embodiment, the CAR T-celltargets CAIX or G250/CAIX, and has a therapeutic effect on subjects withrenal cell carcinoma. In another embodiment, the CAR T-cell targetsCD20, and has a therapeutic effect on subjects with Lymphomas, B-cellmalignancies, B-cell lymphomas, Mantle cell lymphoma and, indolentB-cell lymphomas. In another embodiment, the CAR T-cell targets CD23,and has a therapeutic effect on subjects with CLL. In anotherembodiment, the CAR T-cell targets CD24, and has a therapeutic effect onsubjects with pancreatic adenocarcinoma. In another embodiment, the CART-cell targets CD30, and has a therapeutic effect on subjects withLymphomas or Hodgkin lymphoma. In another embodiment, the CAR T-celltargets CD33, and has a therapeutic effect on subjects with AML. Inanother embodiment, the CAR T-cell targets CD38, and has a therapeuticeffect on subjects with Non-Hodgkin lymphoma. In another embodiment, theCAR T-cell targets CD44v6, and has a therapeutic effect on subjects withseveral malignancies. In another embodiment, the CAR T-cell targetsCD44v7/8, and has a therapeutic effect on subjects with cervicalcarcinoma. In another embodiment, the CAR T-cell targets CD123, and hasa therapeutic effect on subjects with myeloid malignancies. In anotherembodiment, the CAR T-cell targets CEA, and has a therapeutic effect onsubjects with colorectal cancer. In another embodiment, the CAR T-celltargets EGFRvII, and has a therapeutic effect on subjects withGlioblastoma. In another embodiment, the CAR T-cell targets EGP-2, andhas a therapeutic effect on subjects with multiple malignancies. Inanother embodiment, the CAR T-cell targets EGP-40, and has a therapeuticeffect on subjects with colorectal cancer. In another embodiment, theCAR T-cell targets EphA2, and has a therapeutic effect on subjects withGlioblastoma. In another embodiment, the CAR T-cell targets Erb-B2 orErbB3/4, and has a therapeutic effect on subjects with Breast cancer andothers, prostate cancer, colon cancer, various tumors. In anotherembodiment, the CAR T-cell targets Erb-B 2, 3, 4, and has a therapeuticeffect on subjects with Breast cancer and others. In another embodiment,the CAR T-cell targets FBP, and has a therapeutic effect on subjectswith Ovarian cancer. In another embodiment, the CAR T-cell targets fetalacetylcholine receptor, and has a therapeutic effect on subjects withRhabdomyosarcoma. In another embodiment, the CAR T-cell targets G_(D2),and has a therapeutic effect on subjects with Neuroblastoma, melanoma,or Ewing's sarcoma. In another embodiment, the CAR T-cell targetsG_(D3), and has a therapeutic effect on subjects with Melanoma. Inanother embodiment, the CAR T-cell targets HER2, and has a therapeuticeffect on subjects with medulloblastoma, pancreatic adenocarcinoma,Glioblastoma, Osteosarcoma, or Ovarian cancer. In another embodiment,the CAR T-cell targets HMW-MAA, and has a therapeutic effect on subjectswith Melanoma. In another embodiment, the CAR T-cell targetsIL-11Ralpha, and has a therapeutic effect on subjects with Osteosarcoma.In another embodiment, the CAR T-cell targets IL-13Ralpha1, and has atherapeutic effect on subjects with Glioma, Glioblastoma, ormedulloblastoma. In another embodiment, the CAR T-cell targets IL-13receptor alpha2, and has a therapeutic effect on subjects with severalmalignancies. In another embodiment, the CAR T-cell targets KDR, and hasa therapeutic effect on subjects with tumors by targeting tumorneovasculature. In another embodiment, the CAR T-cell targetskappa-light chain, and has a therapeutic effect on subjects with B-cellmalignancies (B-NHL, CLL). In another embodiment, the CAR T-cell targetsLewis Y, and has a therapeutic effect on subjects with variouscarcinomas or epithelial-derived tumors. In another embodiment, the CART-cell targets L1-cell adhesion molecule, and has a therapeutic effecton subjects with Neuroblastoma. In another embodiment, the CAR T-celltargets MAGE-A1 or HLA-A1 MAGE A1, and has a therapeutic effect onsubjects with Melanoma. In another embodiment, the CAR T-cell targetsmesothelin, and has a therapeutic effect on subjects with Mesothelioma.In another embodiment, the CAR T-cell targets CMV infected cells, andhas a therapeutic effect on subjects with CMV. In another embodiment,the CAR T-cell targets MUC1, and has a therapeutic effect on subjectswith breast or ovarian cancer. In another embodiment, the CAR T-celltargets MUC16, and has a therapeutic effect on subjects with ovariancancer. In another embodiment, the CAR T-cell targets NKG2D ligands, andhas a therapeutic effect on subjects with myeloma, ovarian, and othertumors. In another embodiment, the CAR T-cell targets NY-ESO-1 (157-165)or HLA-A2 NY-ESO-1, and has a therapeutic effect on subjects withmultiple myeloma. In another embodiment, the CAR T-cell targetsoncofetal antigen (h5T4), and has a therapeutic effect on subjects withvarious tumors. In another embodiment, the CAR T-cell targets PSCA, andhas a therapeutic effect on subjects with prostate carcinoma. In anotherembodiment, the CAR T-cell targets PSMA, and has a therapeutic effect onsubjects with prostate cancer/tumor vasculature. In another embodiment,the CAR T-cell targets ROR1, and has a therapeutic effect on subjectswith B-CLL and mantle cell lymphoma. In another embodiment, the CART-cell targets TAG-72, and has a therapeutic effect on subjects withadenocarcinomas or gastrointestinal cancers. In another embodiment, theCAR T-cell targets VEGF-R2 or other VEGF receptors, and has atherapeutic effect on subjects with tumors by targeting tumorneovasculature. In another embodiment, the CAR T-cell targets CA9, andhas a therapeutic effect on subjects with renal cell carcinoma. Inanother embodiment, the CAR T-cell targets CD171, and has a therapeuticeffect on subjects with renal neuroblastoma. In another embodiment, theCAR T-cell targets NCAM, and has a therapeutic effect on subjects withneuroblastoma. In another embodiment, the CAR T-cell targets fetalacetylcholine receptor, and has a therapeutic effect on subjects withrhabdomyosarcoma. In another embodiment, the CAR binds to one of thetarget antigens listed in Table 1 of Sadelain et al. (Cancer Discov.2013 April; 3(4): 388-398), which is incorporated by reference herein inits entirety. In another embodiment, CAR T-cells bind to carbohydrate orglycolipid structures.

In one embodiment the CAR binds to an angiogenic factor, therebytargeting tumor vasculature. In one embodiment, the angiogenic factor isVEGFR2. in another embodiment, the angiogenic factor is endoglin. Inanother embodiment, an angiogenic factor disclosed herein is Angiogenin;Angiopoietin-1; Del-1; Fibroblast growth factors: acidic (aFGF) andbasic (bFGF); Follistatin; Granulocyte colony-stimulating factor(G-CSF); Hepatocyte growth factor (HGF)/scatter factor (SF);Interleukin-8 (IL-8); Leptin; Midkine; Placental growth factor;Platelet-derived endothelial cell growth factor (PD-ECGF);Platelet-derived growth factor-BB (PDGF-BB); Pleiotrophin (PTN);Progranulin; Proliferin; Transforming growth factor-alpha (TGF-alpha);Transforming growth factor-beta (TGF-beta); Tumor necrosis factor-alpha(TNF-alpha); Vascular endothelial growth factor (VEGF)/vascularpermeability factor (VPF). In another embodiment, an angiogenic factoris an angiogenic protein. In one embodiment, a growth factor is anangiogenic protein. In one embodiment, an angiogenic protein for use inthe compositions and methods disclosed herein is Fibroblast growthfactors (FGF); VEGF; VEGFR and Neuropilin 1 (NRP-1); Angiopoietin 1(Ang1) and Tie2; Platelet-derived growth factor (PDGF; BB-homodimer) andPDGFR; Transforming growth factor-beta (TGF-β), endoglin and TGF-βreceptors; monocyte chemotactic protein-1 (MCP-1); Integrins αVβ3, αVβ5and α5β1; VE-cadherin and CD31; ephrin; plasminogen activators;plasminogen activator inhibitor-1; Nitric oxide synthase (NOS) andCOX-2; AC133; or Id1/Id3. In one embodiment, an angiogenic protein foruse in the compositions and methods disclosed herein is an angiopoietin,which in one embodiment, is Angiopoietin 1, Angiopoietin 3, Angiopoietin4 or Angiopoietin 6. In one embodiment, endoglin is also known as CD105;EDG; HHT1; ORW; or ORW1. In one embodiment, endoglin is a TGFbetaco-receptor.

In another embodiment, the CAR T-cells bind to an antigen associatedwith an infectious agent. In one embodiment, the infectious agent isMycobacterium tuberculosis. In one embodiment, said Mycobacteriumtuberculosis associated antigen is: Antigen 85B, Lipoprotein IpqH, ATPdependent helicase putative, uncharacterized protein Rv0476/MTO4941precursor or uncharacterized protein Rv1334/MT1376 precursor.

In another embodiment, the CAR binds to an antibody. In one embodiment,the CAR T-cell is an “antibody-coupled T-cell receptor” (ACTR).According to this embodiment, the CAR T-cell is a universal CAR T-cell.In another embodiment, the CAR T-cell having an antibody receptor isadministered before, after, or at the same time as the antibody isadministered and then binds to the antibody, bringing the T-cell inclose proximity to the tumor or cancer. In another embodiment, theantibody is directed against a tumor cell antigen. In anotherembodiment, the antibody is directed against CD20. In anotherembodiment, the antibody is rituximab.

In another embodiment, the antibody is Trastuzumab (Herceptin;Genentech): humanized IgG1, which is directed against ERBB2. In anotherembodiment, the antibody is Bevacizumab (Avastin; Genentech/Roche):humanized IgG1, which is directed against VEGF. In another embodiment,the antibody is Cetuximab (Erbitux; Bristol-Myers Squibb): chimerichuman-murine IgG1, which is directed against EGFR. In anotherembodiment, the antibody is Panitumumab (Vectibix; Amgen): human IgG2,which is directed against EGFR. In another embodiment, the antibody isIpilimumab (Yervoy; Bristol-Myers Squibb): IgG1, which is directedagainst CTLA4.

In another embodiment, the antibody is Alemtuzumab (Campath; Genzyme):humanized IgG1, which is directed against CD52. In another embodiment,the antibody is Ofatumumab (Arzerra; Genmab): human IgG1, which isdirected against CD20. In another embodiment, the antibody is Gemtuzumabozogamicin (Mylotarg; Wyeth): humanized IgG4, which is directed againstCD33. In another embodiment, the antibody is Brentuximab vedotin(Adcetris; Seattle Genetics): chimeric IgG1, which is directed againstCD30. In another embodiment, the antibody is 90Y-labelled ibritumomabtiuxetan (Zevalin; IDEC Pharmaceuticals): murine IgG1, which is directedagainst CD20. In another embodiment, the antibody is 131I-labelledtositumomab (Bexxar; GlaxoSmithKline): murine IgG2, which is directedagainst CD20.

In another embodiment, the antibody is Ramucirumab, which is directedagainst vascular endothelial growth factor receptor-2 (VEGFR-2). Inanother embodiment, the antibody is ramucirumab (Cyramza Injection, EliLilly and Company), blinatumomab (BLINCYTO, Amgen Inc.), pembrolizumab(KEYTRUDA, Merck Sharp & Dohme Corp.), obinutuzumab (GAZYVA, Genentech,Inc.; previously known as GA101), pertuzumab injection (PERJETA,Genentech, Inc.), or denosumab (Xgeva, Amgen Inc.). In anotherembodiment, the antibody is Basiliximab (Simulect; Novartis). In anotherembodiment, the antibody is Daclizumab (Zenapax; Roche).

In another embodiment, the antibody to which the CAR T-cell is coupledis directed to a tumor or cancer antigen or a portion thereof, that isdescribed herein and/or that is known in the art. In another embodiment,the antibody to which the CAR T-cell is couples is directed to atumor-associated antigen. In another embodiment, the antibody to whichthe CAR T-cell is couples is directed to a tumor-associated antigen or aportion thereof that is an angiogenic factor.

In another embodiment, the antibody to which the CAR T-cell is coupledis directed to a tumor or cancer antigen or a portion thereof, that isdescribed herein and/or that is known in the art.

Cytokine Storm and Cytokine Release Syndrome

In one embodiment, a method as disclosed herein includes providingimmune cells, such as NK cells or T-cells comprising engineered chimericantigen receptors, with at least an additional agent to decrease toxiccytokine release or “cytokine release syndrome” (CRS) or “severecytokine release syndrome” (sCRS) or “cytokine storm” that may occur inthe subject. In another embodiment the CRS, sCRS or cytokine stormoccurs as a result of administration of the immune cells. In anotherembodiment, the CRS, sCRS or cytokine storm is the result of a stimulus,condition, or syndrome separate from the immune cells (see below). Inanother embodiment, a cytokine storm, cytokine cascade, orhypercytokinemia is a more severe form of cytokine release syndrome.

In one embodiment, the additional agent for decreasing harmful cytokinerelease comprises apoptotic cells or a composition comprising saidapoptotic cells. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises an apoptotic cellsupernatant or a composition comprising said supernatant. In anotherembodiment, the additional agent for decreasing harmful cytokine releasecomprises a CTLA-4 blocking agent. In another embodiment, the additionalagent for decreasing harmful cytokine release comprises apoptotic cellsor apoptotic cell supernatants or compositions thereof, and a CTLA-4blocking agent. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises an alpha-1 anti-trypsin orfragment thereof or analogue thereof. In another embodiment, theadditional agent for decreasing harmful cytokine release comprisesapoptotic cells or apoptotic cell supernatants or compositions thereof,and an alpha-1 anti-trypsin or fragment thereof or analogue thereof. Inanother embodiment, the additional agent for decreasing harmful cytokinerelease comprises a tellurium-based compound. In another embodiment, theadditional agent for decreasing harmful cytokine release comprisesapoptotic cells or apoptotic cell supernatants or compositions thereof,and a tellurium-based compound. In another embodiment, the additionalagent for decreasing harmful cytokine release comprises an immunemodulating agent. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises apoptotic cells orapoptotic cell supernatants or compositions thereof, and an immunemodulating agent.

A skilled artisan would appreciate that decreasing toxic cytokinerelease or toxic cytokine levels comprises decreasing or inhibitingproduction of toxic cytokine levels in a subject, or inhibiting orreducing the incidence of cytokine release syndrome or a cytokine stormin a subject. In another embodiment toxic cytokine levels are reducedduring CRS or a cytokine storm. In another embodiment, decreasing orinhibiting the production of toxic cytokine levels comprises treatingCRS or a cytokine storm. In another embodiment, decreasing or inhibitingthe production of toxic cytokine levels comprises preventing CRS or acytokine storm. In another embodiment, decreasing or inhibiting theproduction of toxic cytokine levels comprises alleviating CRS or acytokine storm. In another embodiment, decreasing or inhibiting theproduction of toxic cytokine levels comprises ameliorating CRS or acytokine storm. In another embodiment, the toxic cytokines comprisepro-inflammatory cytokines. In another embodiment, pro-inflammatorycytokines comprise IL-6. In another embodiment, pro-inflammatorycytokines comprise IL-1β. In another embodiment, pro-inflammatorycytokines comprise TNF-α, In another embodiment, pro-inflammatorycytokines comprise IL-6, IL-1β, or TNF-α, or any combination thereof.

In one embodiment, cytokine release syndrome is characterized byelevated levels of several inflammatory cytokines and adverse physicalreactions in a subject such as low blood pressure, high fever andshivering. In another embodiment, inflammatory cytokines comprise IL-6,IL-1β, and TNF-α. In another embodiment, CRS is characterized byelevated levels of IL-6, IL-1β, or TNF-α, or any combination thereof. Inanother embodiment, CRS is characterized by elevated levels of IL-8, orIL-13, or any combination thereof. In another embodiment, a cytokinestorm is characterized by increases in TNF-alpha, IFN-gamma, IL-1beta,IL-2, IL-6, IL-8, IL-10, IL-13, GM-CSF, IL-5, fracktalkine, or acombination thereof or a subset thereof. In yet another embodiment, IL-6comprises a marker of CRS or cytokine storm. In another embodiment,IFN-γ comprises a marker of CRS or cytokine storm. In anotherembodiment, patients with larger tumor burdens have higher incidence andseverity of cytokine release syndrome.

In another embodiment, cytokines increased in CRS or a cytokine storm inhumans and mice may comprise any combination of cytokines listed inTables 1 and 2 below.

TABLE 1 Panel of Cytokines Increased in CRS or Cytokine Storm in Humansand/or Mice Mouse model (pre-clinical) CAR-T (H) Mouse Not CytokineHuman origin origin specified Cells secreting this Notes/ Flt-3L * DC(?) Fractalkine * APC, Endothelial cells (?) =CX3CL1, Neurotactin(Mouse) M-CSF =CSF1 GM-CSF * * (in vitro) T cell, MØ IFN-α * T cell, MØ,Monocyte IFN-β ? ? T cell, MØ, Monocyte IFN-γ * * * (in vitro) cytotoxicT cells, helper T cells, NK cells, MØ, Monocyte, DC IL- 1 α * Monocyte,MØ, Epithel IL- 1 β * * Macrophages, DCs, fibroblasts, endothelialcells, hepatocytes IL- 1 Rα * IL- 2 * * * (in vitro) T cells IL- 2Rα *lymphocytes IL- 4 * * * (in vitro) Th2 cells IL- 5 * * * T cells IL-6 * * * monocytes/macrophages, dendritic cells, T cells, fibroblasts,keratinocytes, endothelial cells, adipocytes, myocytes, mesangial cells,and osteoblasts IL- 7 * * In vitro by BM stromal cells IL- 8 *Macrophages, monocytes IL - 9 * * T cells, T helper IL- 10 * * * * (invitro) monocytes/macrophages, mast cells, B cells, regulatory T cells,and helper T cells IL- 12 * * MØ, Monocyte, DC, =p70 activatedlymphocytes, (p40 + p35) neutrophils IL- 13 * * T cells

In one embodiment, cytokines Flt-3L, Fractalkine, GM-CSF, IFN-γ, IL-1β,IL-2, IL-2Rα, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, andIL-13 of Table 1 are considered to be significant in CRS or cytokinestorm. In another embodiment, IFN-α, IFN-β, IL-1, and IL-1Rα of Table 1appear to be important in CRS or cytokine storm. In another embodiment,M-CSF has unknown importance. In another embodiment, any cytokine listedin Table 1, or combination thereof, may be used as a marker of CRS orcytokine storm.

TABLE 2 Panel of Cytokines Increased in CRS or Cytokine Storm in Humansand/or Mice Human model Mouse model (pre-clinical) Cytokine (clinicalCAR-T (H) Mouse Not Cells secreting this Notes/ (Analyte) trials) originorigin specified cytokine other IL- 15 * * Fibroblasts, monocytes (?) 22IL- 17 * * T cells IL- 18 Macrophages IL- 21 * T helper cells, NK cellsIL- 22 * activated DC and T cells IL- 23 IL- 25 Protective? IL- 27 * APCIP-10 * Monocytes (?) MCP-1 * Endothel, fibroblast, =CXCL10 epithel,monocytes MCP-3 * PBMCs, MØ (?) =CCL2 MIP-1α * * (in vitro) T cells=CXCL9 MIP-1β * T cells =CCL3 PAF ? platelets, endothelial cells, =CCL4neutrophils, monocytes, and macrophages, mesangial cells PGE2 * *Gastrointestinal mucosa and other RANTES * Monocytes TGF-β * * MØ,lymphocytes, =CCL5 endothel, platelets . . . TNF-α * * * * (in vitro)Macrophages, NK cells, T cells TNF-αR1 * HGF MIG * T cellchemoattractant, induced by IFN-γ

In one embodiment, IL-15, IL-17, IL-18, IL-21, IL-22, IP-10, MCP-1,MIP-1α, MIP-1β, and TNF-α of Table 2 are considered to be significant inCRS or cytokine storm. In another embodiment, IL-27, MCP-3, PGE2,RANTES, TGF-β, TNF-αR1, and MIG of Table 2 appear to be important in CRSor cytokine storm. In another embodiment, IL-23 and IL-25 have unknownimportance. In another embodiment, any cytokine listed in Table 2, orcombination thereof, may be used as a marker of CRS or cytokine storm.

A skilled artisan would appreciate that the term “cytokine” mayencompass cytokines (e.g., interferon gamma, granulocyte macrophagecolony stimulating factor, tumor necrosis factor alpha), chemokines(e.g., MIP 1 alpha, MIP 1 beta, RANTES), and other soluble mediators ofinflammation, such as reactive oxygen species and nitric oxide.

In one embodiment, increased release of a particular cytokine, whethersignificant, important or having unknown importance, does not a priorimean that the particular cytokine is part of a cytokine storm. In oneembodiment, an increase of at least one cytokine is not the result of acytokine storm or CRS. In another embodiment, CAR T-cells may be thesource of increased levels of a particular cytokine or group ofcytokines.

In another embodiment, cytokine release syndrome is characterized by anyor all of the following symptoms: Fever with or without rigors, malaise,fatigue, anorexia, myalgias, arthalgias, nausea, vomiting, headache SkinRash, Nausea, vomiting, diarrhea, Tachypnea, hypoxemia CardiovascularTachycardia, widened pulse pressure, hypotension, increased cardiacoutput (early), potentially diminished cardiac output (late), ElevatedD-dimer, hypofibrinogenemia with or without bleeding, Azotemia HepaticTransaminitis, hyperbilirubinemia, Headache, mental status changes,confusion, delirium, word finding difficulty or frank aphasia,hallucinations, tremor, dymetria, altered gait, seizures. In anotherembodiment, a cytokine storm is characterized by IL-2 release andlymphoproliferation. In another embodiment, a cytokine storm ischaracterized by increases in cytokines released by CAR T-cells. Inanother embodiment, a cytokine storm is characterized by increases incytokines released by cells other than CAR T-cells.

In another embodiment, cytokine storm leads to potentiallylife-threatening complications including cardiac dysfunction, adultrespiratory distress syndrome, neurologic toxicity, renal and/or hepaticfailure, and disseminated intravascular coagulation.

A skilled artisan would appreciate that the characteristics of acytokine release syndrome (CRS) or cytokine storm are estimated to occura few days to several weeks following the trigger for the CRS orcytokine storm. In one embodiment, CAR T-cells are a trigger for CRS ora cytokine storm. In another embodiment, a trigger for CRS or a cytokinestorm is not CAR T-cells.

In one embodiment, measurement of cytokine levels or concentration, asan indicator of cytokine storm, may be expressed as −fold increase,percent (%) increase, net increase or rate of change in cytokine levelsor concentration. In another embodiment, absolute cytokine levels orconcentrations above a certain level or concentration may be anindication of a subject undergoing or about to experience a cytokinestorm. In another embodiment, absolute cytokine levels or concentrationat a certain level or concentration, for example a level orconcentration normally found in a control subject not undergoing CAR-Tcell therapy, may be an indication of a method for inhibiting orreducing the incidence of a cytokine storm in a subject undergoing CART-cell.

A skilled artisan would appreciate that the term “cytokine level” mayencompass a measure of concentration, a measure of fold change, ameasure of percent (%) change, or a measure of rate change. Further, themethods for measuring cytokines in blood, saliva, serum, urine, andplasma are well known in the art.

In one embodiment, despite the recognition that cytokine storm isassociated with elevation of several inflammatory cytokines, IL-6 levelsmay be used as a common measure of cytokine storm and/or as a commonmeasure of the effectiveness of a treatment for cytokine storms. Askilled artisan would appreciate that other cytokines may be used asmarkers of a cytokine storm, for example any of TNF-α, IB-1α, IL-6,IL-8, IL-13, or INF-γ, or any combination above may be used as a markerof CRS or a cytokine storm. Further, that assay methods for measuringcytokines are well known in the art. A skilled artisan would appreciatethat methods affecting a cytokine storm may similarly affect cytokinerelease syndrome (CRS).

In one embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or a cytokine storm. In another embodiment, disclosedherein is a method of decreasing or inhibiting cytokine production in asubject vulnerable to experiencing cytokine release syndrome or acytokine storm. In another embodiment, methods disclosed herein decreaseor inhibit cytokine production in a subject experiencing cytokinerelease syndrome or a cytokine storm, wherein production of any cytokineor group of cytokines listed in Tables 1 and/or 2 is decreased orinhibited. In another embodiment, cytokine IL-6 production is decreasedor inhibited. In another embodiment, cytokine IL-beta1 production isdecreased or inhibited. In another embodiment, cytokine IL-8 productionis decreased or inhibited. In another embodiment, cytokine IL-13production is decreased or inhibited. In another embodiment, cytokineTNF-alpha production is decreased or inhibited. In another embodiment,cytokines IL-6 production, IL-1beta production, or TNF-alpha production,or any combination thereof is decreased or inhibited.

In one embodiment, cytokine release syndrome is graded. In anotherembodiment, Grade 1 describes cytokine release syndrome in whichsymptoms are not life threatening and require symptomatic treatmentonly, e.g., fever, nausea, fatigue, headache, myalgias, malaise. Inanother embodiment, Grade 2 symptoms require and respond to moderateintervention, such as oxygen, fluids or vasopressor for hypotension. Inanother embodiment, Grade 3 symptoms require and respond to aggressiveintervention. In another embodiment, Grade 4 symptoms arelife-threatening symptoms and require ventilator and patients displayorgan toxicity.

In another embodiment, a cytokine storm is characterized by IL-6 andinterferon gamma release. In another embodiment, a cytokine storm ischaracterized by IL-6 release. In another embodiment, a cytokine stormis characterized by interferon gamma release. In another embodiment, acytokine storm is characterized by release of any cytokine orcombination thereof, listed in Tables 1 and 2. In another embodiment, acytokine storm is characterized by release of any cytokine orcombination thereof, known in the art.

In one embodiment, symptoms onset begins minutes to hours after theinfusion begins. In another embodiment, symptoms coincide with peakcytokine levels.

In one embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy comprises administering anapoptotic cell population or an apoptotic cell supernatant orcompositions thereof. In another embodiment, the apoptotic cellpopulation or an apoptotic cell supernatant or compositions thereof mayaid the CAR T-cell therapy. In another embodiment, the apoptotic cellpopulation or an apoptotic cell supernatant or compositions thereof mayaid in the inhibition or reducing the incidence of the CRS or cytokinestorm. In another embodiment, the apoptotic cell population or anapoptotic cell supernatant or compositions thereof may aid in treatingthe CRS or cytokine storm. In another embodiment, the apoptotic cellpopulation or an apoptotic cell supernatant or compositions thereof mayaid in preventing the CRS or cytokine storm. In another embodiment, theapoptotic cell population or an apoptotic cell supernatant orcompositions thereof may aid in ameliorating the CRS or cytokine storm.In another embodiment, the apoptotic cell population or an apoptoticcell supernatant or compositions thereof may aid in alleviating the CRSor cytokine storm.

In one embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy comprises administering anadditional agent. In another embodiment, the additional agent may aidthe CAR T-cell therapy. In one embodiment, a method of inhibiting orreducing the incidence of a cytokine release syndrome (CRS) or acytokine storm in a subject undergoing TCR T-cell cancer therapycomprises administering an additional agent. In another embodiment, theadditional agent may aid the TCR T-cell therapy. In one embodiment, amethod of inhibiting or reducing the incidence of a cytokine releasesyndrome (CRS) or a cytokine storm in a subject undergoing dendriticcell therapy comprises administering an additional agent. In anotherembodiment, the additional agent may aid the dendritic cell therapy. Inone embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing NK cell therapy comprises administering an additional agent.In another embodiment, the additional agent may aid the NK cell therapy.

In one embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy, and being administered anapoptotic cell population or an apoptotic cell supernatant orcompositions thereof, comprises administering an additional agent. Inanother embodiment, the additional agent may aid the CAR T-cell therapy.In another embodiment, the additional agent may aid in the inhibition orreducing the incidence of the CRS or cytokine storm. In anotherembodiment, the additional agent may aid in treating the CRS or cytokinestorm. In another embodiment, the additional agent may aid in preventingthe CRS or cytokine storm. In another embodiment, the additional agentmay aid in ameliorating the CRS or cytokine storm. In anotherembodiment, the additional agent may aid in alleviating the CRS orcytokine storm.

In one embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy comprises administering anadditional agent. In another embodiment, the additional agent may aidthe CAR T-cell therapy. In another embodiment, the additional agent mayaid in the inhibition or reducing the incidence of the CRS or cytokinestorm. In another embodiment, the additional agent may aid in treatingthe CRS or cytokine storm. In another embodiment, the additional agentmay aid in preventing the CRS or cytokine storm. In another embodiment,the additional agent may aid in ameliorating the CRS or cytokine storm.In another embodiment, the additional agent may aid in alleviating theCRS or cytokine storm.

In one embodiment, the additional agent for decreasing harmful cytokinerelease comprises apoptotic cells or a composition comprising saidapoptotic cells. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises an apoptotic cellsupernatant or a composition comprising said supernatant. In anotherembodiment, the additional agent for decreasing harmful cytokine releasecomprises a CTLA-4 blocking agent. In another embodiment, the additionalagent for decreasing harmful cytokine release comprises apoptotic cellsor apoptotic cell supernatants or compositions thereof, and a CTLA-4blocking agent. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises an alpha-1 anti-trypsin orfragment thereof or analogue thereof. In another embodiment, theadditional agent for decreasing harmful cytokine release comprisesapoptotic cells or apoptotic cell supernatants or compositions thereof,and an alpha-1 anti-trypsin or fragment thereof or analogue thereof. Inanother embodiment, the additional agent for decreasing harmful cytokinerelease comprises a tellurium-based compound. In another embodiment, theadditional agent for decreasing harmful cytokine release comprisesapoptotic cells or apoptotic cell supernatants or compositions thereof,and a tellurium-based compound. In another embodiment, the additionalagent for decreasing harmful cytokine release comprises an immunemodulating agent. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises apoptotic cells orapoptotic cell supernatants or compositions thereof, and an immunemodulating agent. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises Treg cells. In anotherembodiment, the additional agent for decreasing harmful cytokine releasecomprises apoptotic cells or apoptotic cell supernatants or compositionsthereof, and Treg cells.

In another embodiment, compositions and methods as disclosed hereinutilize combination therapy of CAR T-cells with one or moreCTLA-4-blocking agents such as Ipilimumab. In another embodiment, CTLA-4is a potent inhibitor of T-cell activation that helps to maintainself-tolerance. In another embodiment, administration of an anti-CTLA-4blocking agent, which in another embodiment, is an antibody, produces anet effect of T-cell activation. In another embodiment, compositions andmethods as disclosed herein utilize combined therapy comprisingapoptotic cells, CAR T-cells, and one or more CTLA-4-blocking agents.

In another embodiment, other toxicities resulting from CAR T-cell or NKcell administration that may be treated, prevented, inhibited,ameliorated, reduced in incidence or alleviated by the compositions andmethods as disclosed herein comprise B cell aplasia or tumor lysissyndrome (TLS).

In one embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy does not affect the efficacy of theCAR T-cell therapy. In another embodiment, a method of inhibiting orreducing the incidence of CRS or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy, does reduce the efficacy of theCAR T-cells therapy by more than about 5%. In another embodiment, amethod of inhibiting or reducing the incidence of CRS or a cytokinestorm in a subject undergoing CAR T-cell cancer therapy, does reduce theefficacy of the CAR T-cells therapy by more than about 10%. In anotherembodiment, a method of inhibiting or reducing the incidence of CRS or acytokine storm in a subject undergoing CAR T-cell cancer therapy, doesreduce the efficacy of the CAR T-cells therapy by more than about 15%.In another embodiment, a method of inhibiting or reducing the incidenceof CRS or a cytokine storm in a subject undergoing CAR T-cell cancertherapy, does reduce the efficacy of the CAR T-cells therapy by morethan about 20%.

Any appropriate method of quantifying cytotoxicity can be used todetermine whether activity in an immune cell modified to express a CARremains substantially unchanged. For example, cytotoxicity can bequantified using a cell culture-based assay such as the cytotoxic assaysdescribed in the Examples. Cytotoxicity assays can employ dyes thatpreferentially stain the DNA of dead cells. In other cases, fluorescentand luminescent assays that measure the relative number of live and deadcells in a cell population can be used. For such assays, proteaseactivities serve as markers for cell viability and cell toxicity, and alabeled cell permeable peptide generates fluorescent signals that areproportional to the number of viable cells in the sample. Kits forvarious cytotoxicity assays are commercially available frommanufacturers such as Promega and Life Technologies. In anotherembodiment, a measure of cytotoxicity may be qualitative. In anotherembodiment, a measure of cytotoxicity may be quantitative. In a furtherembodiment a measure of cytotoxicity may be related to the change inexpression of a cytotoxic cytokine.

In one embodiment, the methods as disclosed herein comprise anadditional step that is useful in overcoming rejection of allogeneicdonor cells. In one embodiment, the methods comprise the step of full orpartial lymphodepletion prior to administration of the CAR T-cells,which in one embodiment, are allogeneic CAR T-cells. In anotherembodiment, the lymphodepletion is adjusted so that it delays the hostversus graft reaction for a period sufficient to allow said allogeneicT-cells to attack the tumor to which they are directed, but to an extentinsufficient to require rescue of the host immune system by bone marrowtransplantation. In another embodiment, agents that delay egression ofthe allogeneic T-cells from lymph nodes, such as2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol (FTY720),5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)pheny-1]1,2,4-oxadiazole(SEW2871), 3-(2-(-hexylphenylamino)-2-oxoethylamino)propanoic acid(W123),2-ammonio-4-(2-chloro-4-(3-phenoxyphenylthio)phenyl)-2-(hydroxymethyl)but-ylhydrogen phosphate (KRP-203 phosphate) or other agents known in the art,may be used as part of the compositions and methods as disclosed hereinto allow the use of allogeneic CAR T-cells having efficacy and lackinginitiation of graft vs host disease. In one embodiment, MHC expressionby the allogeneic T-cells is silenced to reduce the rejection of theallogeneic cells. In another embodiment, the apoptotic cells preventrejection of the allogeneic cells.

Cytokine Release Associated with CAR T-Cell Therapy

In one embodiment, cytokine release occurs between a few days to 2 weeksafter administration of immune therapy such as CAR T-cell therapy. Inone embodiment, hypotension and other symptoms follow the cytokinerelease, i.e. from few days to few weeks. Therefore, in one embodiment,apoptotic cells or the apoptotic cell supernatant are administered tosubjects at the same time as immune therapy as prophylaxis. In anotherembodiment, apoptotic cells or supernatant are administered to subjects2-3 days after administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 7 days afteradministration of immune therapy. In another embodiment, apoptotic cellsor supernatant are administered to subjects 10 days after administrationof immune therapy. In another embodiment, apoptotic cells or supernatantare administered to subjects 14 days after administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 2-14 days after administration of immunetherapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant areadministered to subjects 2-3 hours after administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 7 hours after administration of immune therapy.In another embodiment, apoptotic cells or supernatant are administeredto subjects 10 hours after administration of immune therapy. In anotherembodiment, apoptotic cells or supernatant are administered to subjects14 hours after administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 2-14 hoursafter administration of immune therapy.

In an alternative embodiment, apoptotic cells or the apoptotic cellsupernatant are administered to subjects prior to immune therapy asprophylaxis. In another embodiment, apoptotic cells or supernatant areadministered to subjects 1 day before administration of immune therapy.In another embodiment, apoptotic cells or supernatant are administeredto subjects 2-3 days before administration of immune therapy. In anotherembodiment, apoptotic cells or supernatant are administered to subjects7 days before administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 10 daysbefore administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 14 daysbefore administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 2-14 daysbefore administration of immune therapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant areadministered to subjects 2-3 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 7 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 10 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 14 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 2-14 hours before administration of immunetherapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant maybe administered therapeutically, once cytokine release syndrome hasoccurred. In one embodiment, apoptotic cells or supernatant may beadministered once cytokine release leading up to or attesting to thebeginning of cytokine release syndrome is detected. In one embodiment,apoptotic cells or supernatant can terminate the increased cytokinelevels, or the cytokine release syndrome, and avoid its sequelae.

In another embodiment, apoptotic cells or apoptotic cell supernatant maybe administered therapeutically, at multiple time points. In anotherembodiment, administration of apoptotic cells or apoptotic cellsupernatant is at least at two time points described herein. In anotherembodiment, administration of apoptotic cells or apoptotic cellsupernatant is at least at three time points described herein. Inanother embodiment, administration of apoptotic cells or apoptotic cellsupernatant is prior to CRS or a cytokine storm, and once cytokinerelease syndrome has occurred, and any combination thereof.

In one embodiment, the chimeric antigen receptor-expressing T-cell (CART-cell) therapy and the apoptotic cell therapy or supernatant areadministered together. In another embodiment, the CAR T-cell therapy isadministered after the apoptotic cell therapy or supernatant. In anotherembodiment, the CAR T-cell therapy is administered prior to theapoptotic cell therapy or supernatant. According to this aspect and inone embodiment, apoptotic cell therapy or supernatant is administeredapproximately 2-3 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy or supernatant is administeredapproximately 6-7 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy or supernatant is administeredapproximately 9 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy is administered up to several monthsafter CAR T-cell therapy.

Therefore, in one embodiment, apoptotic cells or the apoptotic cellsupernatant are administered to subjects at the same time as immunetherapy as prophylaxis. In another embodiment, apoptotic cells orsupernatant are administered to subjects 2-3 days after administrationof immune therapy. In another embodiment, apoptotic cells or supernatantare administered to subjects 7 days after administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 10 days after administration of immune therapy.In another embodiment, apoptotic cells or supernatant are administeredto subjects 14 days after administration of immune therapy. In anotherembodiment, apoptotic cells or supernatant are administered to subjects2-14 days after administration of immune therapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant areadministered to subjects 2-3 hours after administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 7 hours after administration of immune therapy.In another embodiment, apoptotic cells or supernatant are administeredto subjects 10 hours after administration of immune therapy. In anotherembodiment, apoptotic cells or supernatant are administered to subjects14 hours after administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 2-14 hoursafter administration of immune therapy.

In an alternative embodiment, apoptotic cells or the apoptotic cellsupernatant are administered to subjects prior to immune therapy asprophylaxis. In another embodiment, apoptotic cells or supernatant areadministered to subjects 1 day before administration of immune therapy.In another embodiment, apoptotic cells or supernatant are administeredto subjects 2-3 days before administration of immune therapy. In anotherembodiment, apoptotic cells or supernatant are administered to subjects7 days before administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 10 daysbefore administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 14 daysbefore administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 2-14 daysbefore administration of immune therapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant areadministered to subjects 2-3 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 7 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 10 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 14 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 2-14 hours before administration of immunetherapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant maybe administered therapeutically, once cytokine release syndrome hasoccurred. In one embodiment, apoptotic cells or supernatant may beadministered once cytokine release leading up to or attesting to thebeginning of cytokine release syndrome is detected. In one embodiment,apoptotic cells or supernatant can terminate the increased cytokinelevels, or the cytokine release syndrome, and avoid its sequelae.

In another embodiment, apoptotic cells or apoptotic cell supernatant maybe administered therapeutically, at multiple time points. In anotherembodiment, administration of apoptotic cells or apoptotic cellsupernatant is at least at two time points described herein. In anotherembodiment, administration of apoptotic cells or apoptotic cellsupernatant is at least at three time points described herein. Inanother embodiment, administration of apoptotic cells or apoptotic cellsupernatant is prior to CRS or a cytokine storm, and once cytokinerelease syndrome has occurred, and any combination thereof.

In one embodiment, the chimeric antigen receptor-expressing T-cell (CART-cell) therapy and the apoptotic cell therapy or supernatant areadministered together. In another embodiment, the CAR T-cell therapy isadministered after the apoptotic cell therapy or supernatant. In anotherembodiment, the CAR T-cell therapy is administered prior to theapoptotic cell therapy or supernatant. According to this aspect and inone embodiment, apoptotic cell therapy or supernatant is administeredapproximately 2-3 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy or supernatant is administeredapproximately 6-7 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy or supernatant is administeredapproximately 9 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy is administered up to several monthsafter CAR T-cell therapy.

In other embodiments, an additional agent is administered to subjects atthe same time as immune therapy as prophylaxis. In one embodiment theadditional agent comprises apoptotic cells, an apoptotic supernatant, aCTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment thereof oranalogue thereof, of a tellurium-based compound, or an immune-modulatingcompounds, or any combination thereof. In another embodiment, theadditional agent is administered to subjects 2-3 days afteradministration of immune therapy. In another embodiment, the additionalagent is administered to subjects 7 days after administration of immunetherapy. In another embodiment, the additional agent is administered tosubjects 10 days after administration of immune therapy. In anotherembodiment, the additional agent is administered to subjects 14 daysafter administration of immune therapy. In another embodiment, theadditional agent is administered to subjects 2-14 days afteradministration of immune therapy.

In another embodiment, the additional agent is administered to subjects2-3 hours after administration of immune therapy. In another embodiment,the additional agent is administered to subjects 7 hours afteradministration of immune therapy. In another embodiment the additionalagent is administered to subjects 10 hours after administration ofimmune therapy. In another embodiment, the additional agent isadministered to subjects 14 hours after administration of immunetherapy. In another embodiment, the additional agent is administered tosubjects 2-14 hours after administration of immune therapy.

In an alternative embodiment, the additional agent is administered tosubjects prior to immune therapy as prophylaxis. In another embodiment,the additional agent is administered to subjects 1 day beforeadministration of immune therapy. In another embodiment, the additionalagent is administered to subjects 2-3 days before administration ofimmune therapy. In another embodiment, the additional agent isadministered to subjects 7 days before administration of immune therapy.In another embodiment, the additional agent is administered to subjects10 days before administration of immune therapy. In another embodiment,the additional agent is administered to subjects 14 days beforeadministration of immune therapy. In another embodiment, the additionalagent is administered to subjects 2-14 days before administration ofimmune therapy.

In another embodiment, the additional agent is administered to subjects2-3 hours before administration of immune therapy. In anotherembodiment, the additional agent is administered to subjects 7 hoursbefore administration of immune therapy. In another embodiment, theadditional agent is administered to subjects 10 hours beforeadministration of immune therapy. In another embodiment, the additionalagent is administered to subjects 14 hours before administration ofimmune therapy. In another embodiment, the additional agent isadministered to subjects 2-14 hours before administration of immunetherapy.

In another embodiment, the additional agent is administeredtherapeutically, once cytokine release syndrome has occurred. In oneembodiment, the additional agent is administered once cytokine releaseleading up to or attesting to the beginning of cytokine release syndromeis detected. In one embodiment, the additional agent can terminate theincreased cytokine levels, or the cytokine release syndrome, and avoidits sequelae.

In another embodiment, the additional agent is administeredtherapeutically, at multiple time points. In another embodiment,administration of the additional agent is at least at two time pointsdescribed herein. In another embodiment, administration of theadditional agent is at least at three time points described herein. Inanother embodiment, administration of the additional agent is prior toCRS or a cytokine storm, and once cytokine release syndrome hasoccurred, and any combination thereof.

In one embodiment, the chimeric antigen receptor-expressing T-cell (CART-cell) therapy and the additional agent is administered together. Inanother embodiment, the CAR T-cell therapy is administered theadditional agent. In another embodiment, the CAR T-cell therapy isadministered prior to the additional agent. According to this aspect andin one embodiment, the additional agent is administered approximately2-3 weeks after the CAR T-cell therapy. In another embodiment, theadditional agent is administered approximately 6-7 weeks after the CART-cell therapy. In another embodiment, the additional agent isadministered approximately 9 weeks after the CAR T-cell therapy. Inanother embodiment, the additional agent is administered up to severalmonths after CAR T-cell therapy.

In one embodiment, CAR T-cells are heterologous to the subject. In oneembodiment, CAR T-cells are derived from one or more donors. In oneembodiment, CAR T-cells are derived from one or more bone marrow donors.In another embodiment, CAR T-cells are derived from one or more bloodbank donations. In one embodiment, the donors are matched donors. In oneembodiment, CAR T-cells are universal allogeneic CAR T-cells. In anotherembodiment, CAR T-cells are syngeneic CAR T-cells. In anotherembodiment, CAR T-cells are from unmatched third party donors. Inanother embodiment, CAR T-cells are from pooled third party donorT-cells. In one embodiment, the donor is a bone marrow donor. In anotherembodiment, the donor is a blood bank donor. In one embodiment, CART-cells of the compositions and methods as disclosed herein comprise oneor more MHC unrestricted tumor-directed chimeric receptors. In oneembodiment, non-autologous T-cells may be engineered or administeredaccording to protocols known in the art to prevent or minimizeautoimmune reactions, such as described in U.S. Patent Application No.20130156794, which is incorporated herein by references in its entirety.

In another embodiment, CAR T-cells are autologous to the subject. In oneembodiment, the patient's own cells are used. In this embodiment, if thepatient's own cells are used, then the CAR T-cell therapy isadministered after the apoptotic cell therapy.

In one embodiment, apoptotic cells are heterologous to the subject. Inone embodiment, apoptotic cells are derived from one or more donors. Inone embodiment, apoptotic cells are derived from one or more bone marrowdonors. In another embodiment, apoptotic cells are derived from one ormore blood bank donations. In one embodiment, the donors are matcheddonors. In another embodiment, apoptotic cells are from unmatched thirdparty donors. In one embodiment, apoptotic cells are universalallogeneic apoptotic cells. In another embodiment, apoptotic cells arefrom a syngeneic donor. In another embodiment, apoptotic cells are frompooled third party donor cells. In one embodiment, the donor is a bonemarrow donor. In another embodiment, the donor is a blood bank donor. Inanother embodiment, apoptotic cells are autologous to the subject. Inthis embodiment, the patient's own cells are used.

According to some embodiments, the therapeutic mononuclear-enriched cellpreparation disclosed herein or the apoptotic cell supernatant isadministered to the subject systemically. In another embodiment,administration is via the intravenous route. Alternately, thetherapeutic mononuclear enriched cell or supernatant may be administeredto the subject according to various other routes, including, but notlimited to, the parenteral, intraperitoneal, intra-articular,intramuscular and subcutaneous routes. Each possibility represents aseparate embodiment as disclosed herein.

According to some embodiments, the therapeutic mononuclear-enriched cellpreparation disclosed herein or the additional agent is administered tothe subject systemically. In another embodiment, administration is viathe intravenous route. Alternately, the therapeutic mononuclear enrichedcell or the additional agent may be administered to the subjectaccording to various other routes, including, but not limited to, theparenteral, intraperitoneal, intra-articular, intramuscular andsubcutaneous routes. Each possibility represents a separate embodimentas disclosed herein.

In one embodiment, the preparation is administered in a local ratherthan systemic manner, for example, via injection of the preparationdirectly into a specific region of a patient's body. In anotherembodiment, a specific region comprises a tumor or cancer.

In another embodiment, the therapeutic mononuclear enriched cells orsupernatant are administered to the subject suspended in a suitablephysiological buffer, such as, but not limited to, saline solution, PBS,HBSS, and the like. In addition the suspension medium may furthercomprise supplements conducive to maintaining the viability of thecells. In another embodiment, the additional agent is administered tothe subject suspended in a suitable physiological buffer, such as, butnot limited to, saline solution, PBS, HBSS, and the like.

According to some embodiments the pharmaceutical composition isadministered intravenously. According to another embodiment, thepharmaceutical composition is administered in a single dose. Accordingto alternative embodiments the pharmaceutical composition isadministered in multiple doses. According to another embodiment, thepharmaceutical composition is administered in two doses. According toanother embodiment, the pharmaceutical composition is administered inthree doses. According to another embodiment, the pharmaceuticalcomposition is administered in four doses. According to anotherembodiment, the pharmaceutical composition is administered in five ormore doses. According to some embodiments, the pharmaceuticalcomposition is formulated for intravenous injection.

In one embodiment, any appropriate method of providing modifiedCAR-expressing immune cells to a subject can be used for methodsdescribed herein. In one embodiment, methods for providing cells to asubject comprise hematopoietic cell transplantation (HCT), infusion ofdonor-derived NK cells into cancer patients or a combination thereof.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) therapy, comprising the step of administering a compositioncomprising apoptotic cells to said subject.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) therapy, comprising the step of administering an apoptotic cellsupernatant, such as an apoptotic cell-phagocyte supernatant, to saidsubject.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) therapy, comprising the step of administering an at least oneadditional agent to said subject.

In certain embodiments, a CAR T-cell therapy comprises administering acomposition disclosed herein comprising CAR T-cells and either apoptoticcells or an apoptotic cell supernatant, or another or combination ofadditional agents as disclosed herein. In alternative embodiments, a CART-cell therapy comprises administering a composition disclosed hereincomprising CAR T-cells and a composition comprising either apoptoticcells or an apoptotic cell supernatant, or an additional agent orcombination thereof as disclosed herein.

Cytokine Release Associated with Non CAR T-Cell Applications

In one embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm, comprising the step of administering acomposition comprising apoptotic cells or an apoptotic supernatant tosaid subject, wherein said administering decreases or inhibits cytokineproduction in said subject. In another embodiment, decrease orinhibition of cytokine production is compared with a subjectexperiencing cytokine release syndrome or cytokine storm or vulnerableto cytokine release syndrome or cytokine storm and not administeredapoptotic cells or an apoptotic supernatant. In another embodiment,methods for decreasing or inhibiting cytokine production decrease orinhibit pro-inflammatory cytokine production. In another embodiment,methods for decreasing or inhibiting cytokine production decrease orinhibit production of at least one pro-inflammatory cytokine. In anotherembodiment, methods for decreasing or inhibiting cytokine productiondecrease or inhibit production of at least cytokine IL-6. In anotherembodiment, methods for decreasing or inhibiting cytokine productiondecrease or inhibit production of at least cytokine IL-1beta. In anotherembodiment, methods for decreasing or inhibiting cytokine productiondecrease or inhibit production of at least cytokine TNF-alpha. Inanother embodiment, methods disclosed herein for decreasing orinhibiting cytokine production, result in reduction or inhibition ofproduction of cytokines IL-6, IL-1β, or TNF-α, or any combination insaid subject compared with a subject experiencing cytokine releasesyndrome or cytokine storm or vulnerable to cytokine release syndrome orcytokine storm and not administered apoptotic cells or an apoptoticsupernatant.

Cancers or tumors may also affect the absolute level of cytokinesincluding pro-inflammatory cytokines. The level of tumor burden in asubject may affect cytokine levels, particularly proinflammatorycytokines. A skilled artisan would appreciate that the phrase “decreaseor inhibit” or grammatical variants thereof may encompass fold decreaseor inhibition of cytokine production, or a net decrease or inhibition ofcytokine production, or percent (%) decrease or inhibition, or mayencompass a rate of change of decrease or inhibition of a cytokineproduction.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm comprising the step of administeringapoptotic cells or a composition comprising apoptotic cells to saidsubject.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm comprising the step of administering anapoptotic cell supernatant, such as an apoptotic cell-phagocytesupernatant, or a composition comprising said supernatant to saidsubject.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm comprising the step of administering anapoptotic cell supernatant, such as an additional agent selected fromthe group comprising apoptotic cells, an apoptotic supernatant, a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, a tellurium-based compound, or an immune modulating agent, orany combination thereof, or a composition comprising said supernatant tosaid subject.

In one embodiment, an infection causes the cytokine release syndrome orcytokine storm in the subject. In one embodiment, the infection is aninfluenza infection. In one embodiment, the influenza infection is H1N1.In another embodiment, the influenza infection is an H5N1 bird flu. Inanother embodiment, the infection is severe acute respiratory syndrome(SARS). In another embodiment, the subject has Epstein-Barrvirus-associated hemophagocytic lymphohistiocytosis (HLH). In anotherembodiment, the infection is sepsis. In one embodiment, the sepsis isgram-negative. In another embodiment, the infection is malaria. Inanother embodiment, the infection is an Ebola virus infection. Inanother embodiment, the infection is variola virus. In anotherembodiment, the infection is a systemic Gram-negative bacterialinfection. In another embodiment, the infection is Jarisch-Herxheimersyndrome.

In one embodiment, the cause of the cytokine release syndrome orcytokine storm in a subject is hemophagocytic lymphohistiocytosis (HLH).In another embodiment, HLH is sporadic HLH. In another embodiment, HLHis macrophage activation syndrome (MAS). In another embodiment, thecause of the cytokine release syndrome or cytokine storm in a subject isMAS.

In one embodiment, the cause of the cytokine release syndrome orcytokine storm in a subject is chronic arthritis. In another embodiment,the cause of the cytokine release syndrome or cytokine storm in asubject is systemic Juvenile Idiopathic Arthritis (sJIA), also known asStill's Disease.

In one embodiment, the cause of the cytokine release syndrome orcytokine storm in a subject is Cryopyrin-associated Periodic Syndrome(CAPS). In another embodiment, CAPS comprises Familial ColdAuto-inflammatory Syndrome (FCAS), also known as Familial Cold Urticaria(FCU). In another embodiment, CAPS comprises Muckle-Well Syndrome (MWS).In another embodiment, CAPS comprises Chronic Infantile NeurologicalCutaneous and Articular (CINCA) Syndrome. In yet another embodiment,CAPS comprises FCAS, FCU, MWS, or CINCA Syndrome, or any combinationthereof. In another embodiment, the cause of the cytokine releasesyndrome or cytokine storm in a subject is FCAS. In another embodiment,the cause of the cytokine release syndrome or cytokine storm in asubject is FCU. In another embodiment, the cause of the cytokine releasesyndrome or cytokine storm in a subject is MWS. In another embodiment,the cause of the cytokine release syndrome or cytokine storm in asubject is CINCA Syndrome. In still another embodiment, the cause of thecytokine release syndrome or cytokine storm in a subject is FCAS, FCU,MWS, or CINCA Syndrome, or any combination thereof.

In another embodiment, the cause of the cytokine release syndrome orcytokine storm in a subject is a cryopyrinopathy comprising inherited orde novo gain of function mutations in the NLRP3 gene, also known as theCIASI gene.

In one embodiment, the cause of the cytokine release syndrome orcytokine storm in a subject is a hereditary auto-inflammatory disorder.

In one embodiment, the trigger for the release of inflammatory cytokinesis a lipopolysaccharide (LPS), Gram-positive toxins, fungal toxins,glycosylphosphatidylinositol (GPI) or modulation of RIG-1 geneexpression.

In another embodiment, the subject experiencing cytokine releasesyndrome or cytokine storm does not have an infectious disease. In oneembodiment, the subject has acute pancreatitis. In another embodiment,the subject has tissue injury, which in on embodiment, is severe burnsor trauma. In another embodiment, the subject has acute respiratorydistress syndrome. In another embodiment, the subject has cytokinerelease syndrome or cytokine storm secondary to agent use. In anotherembodiment, the subject has cytokine release syndrome or cytokine stormsecondary to toxin inhalation.

In another embodiment, the subject has cytokine release syndrome orcytokine storm secondary to receipt of immunotherapy, which in oneembodiment is immunotherapy with superagonistic CD28-specific monoclonalantibodies (CD28SA). In one embodiment, the CD28SA is TGN1412. Inanother embodiment, the immunotherapy is CAR T-cell therapy. In anotherembodiment, the immunotherapy is dendritic cell therapy.

In another embodiment, apoptotic cells or supernatant or a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, a tellurium-based compound, or an immune modulating agent, orany combination thereof, may be used to control cytokine releasesyndrome or cytokine storm that results from administration of apharmaceutical composition. In one embodiment, the pharmaceuticalcomposition is oxaliplatin, cytarabine, lenalidomide, or a combinationthereof.

In another embodiment, apoptotic cells or the supernatant or a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, a tellurium-based compound, or an immune modulating agent, orany combination thereof, may be used to control cytokine releasesyndrome or cytokine storm that results from administration of anantibody. In one embodiment, the antibody is monoclonal. In anotherembodiment, the antibody is polyclonal. In one embodiment, the antibodyis rituximab. In another embodiment, the antibody is Orthoclone OKT3(muromonab-CD3). In another embodiment, the antibody is alemtuzumab,tosituzumab, CP-870,893, LO-CD2a/BTI-322 or TGN1412.

In another embodiment, examples of diseases for which control ofinflammatory cytokine production can be beneficial include cancers,allergies, any type of infection, toxic shock syndrome, sepsis, any typeof autoimmune disease, arthritis, Crohn's disease, lupus, psoriasis, orany other disease for which the hallmark feature is toxic cytokinerelease that causes deleterious effects in a subject.

T-Cell Receptors (TCRs)

In another embodiment, compositions and methods as disclosed hereinutilize combination therapy of apoptotic cells or apoptotic cellsupernatants, or a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment thereof or analogue thereof, a tellurium-based compound, or animmune modulating agent, or any combination thereof, and designer T-cellreceptors in addition to or in place of CAR T-cells. In one embodiment,TCR therapy comprises introducing a T-cell receptor (TCR) that isspecific to an epitope of a protein of interest into a T-cell. Inanother embodiment, the protein of interest is a tumor-associatedantigen. In another embodiment, the genetically engineered TCRrecognizes a tumor antigen epitope presented by the majorhistocompatibility complex (MHC) on the tumor cell along with T-cellactivating domains. In another embodiment, the T-cell receptorsrecognize antigens irrespectively of their intracellular or membranelocalization. In another embodiment, TCRs recognize tumor cells thatintracellularly express a tumor associated antigen. In one embodimentTCRs recognize internal antigens. Various genetically modified T-cellreceptors and methods of their production are known in the art.

In one embodiment, TCR therapy is used to treat, prevent, inhibit,ameliorate, reduce the incidence of, or alleviate advanced metastaticdisease, including those with hematological (lymphoma and leukemia) andsolid tumors (refractory melanoma, sarcoma). In another embodiment, theT-cell receptor is genetically modified to bind NY-ESO-1 epitopes, andthe TCR-engineered T-cell is anti-NY-ESO-1. In another embodiment, theT-cell receptor is genetically modified to bind HPV-16 E6 epitopes, andthe TCR-engineered T-cell is anti-HPV-16 E6. In another embodiment, theT-cell receptor is genetically modified to bind HPV-16 E7 epitopes, andthe TCR-engineered T-cell is anti-HPV-16 E7. In another embodiment, theT-cell receptor is genetically modified to bind MAGE A3/A6 epitopes, andthe TCR-engineered T-cell is anti-MAGE A3/A6. In another embodiment, theT-cell receptor is genetically modified to bind MAGE A3 epitopes, andthe TCR-engineered T-cell is anti-MAGE A3. In another embodiment, theT-cell receptor is genetically modified to bind SSX2 epitopes, and theTCR-engineered T-cell is anti-SSX2.

In one embodiment, the TCR therapy used in the compositions and methodsas disclosed herein treat a malignancy listed in Table 1 of Sadelain etal., (ibid).

Dendritic Cells

In another embodiment, apoptotic cells or apoptotic supernatants or aCTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment thereof oranalogue thereof, a tellurium-based compound, or an immune modulatingagent, or any combination thereof, may be used to increase the safety ofimmunotherapy with dendritic cells. In one embodiment, dendritic cells(DCs) are antigen-producing and presenting cells of the mammalian immunesystem that process antigen material and present it on the cell surfaceto the T-cells of the immune system and are thereby capable ofsensitizing T-cells to both new and recall antigens. In anotherembodiment, DCs are the most potent antigen-producing cells, acting asmessengers between the innate and the adaptive immune systems. DC cellsmay be used, in one embodiment, to prime specific antitumor immunitythrough the generation of effector cells that attack and lyse tumors.

Dendritic cells are present in those tissues that are in contact withthe external environment, such as the skin (where there is a specializeddendritic cell type called the Langerhans cell) and the inner lining ofthe nose, lungs, stomach and intestines. They can also be found in animmature state in the blood. Once activated, they migrate to the lymphnodes where they interact with T-cells and B cells to initiate and shapethe adaptive immune response. At certain development stages, they growbranched projections, the dendrites that give the cell its name.Dendritic cells may be engineered to express particular tumor antigens.

The three signals that are required for T-cell activation are: (i)presentation of cognate antigen in self MHC molecules; (ii)costimulation by membrane-bound receptor-ligand pairs; and (iii) solublefactors to direct polarization of the ensuing immune response. Dendriticcells (DCs) are able to provide all of the three signals required forT-cell activation making them an excellent cancer vaccine platform.

Therefore, in another embodiment, disclosed herein are a compositioncomprising dendritic cells and apoptotic cells or apoptotic supernatantsor a CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment thereofor analogue thereof, a tellurium-based compound, or an immune modulatingagent, or any combination thereof.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing dendritic cell therapy, comprising the step ofadministering a composition comprising apoptotic cells or apoptotic cellsupernatants or a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment thereof or analogue thereof, a tellurium-based compound, or animmune modulating agent, or any combination thereof, to said subject. Inanother embodiment, a method of treating cytokine release syndrome orcytokine storm in a subject undergoing dendritic cell therapy, comprisesthe step of administering a composition comprising apoptotic cells orapoptotic cell supernatants or a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment thereof or analogue thereof, a tellurium-basedcompound, or an immune modulating agent, or any combination thereof, tosaid subject. In another embodiment, a method of preventing cytokinerelease syndrome or cytokine storm in a subject undergoing dendriticcell therapy, comprises the step of administering a compositioncomprising apoptotic cells or apoptotic cell supernatants or a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, a tellurium-based compound, or an immune modulating agent, orany combination thereof, to said subject. In another embodiment, amethod of ameliorating cytokine release syndrome or cytokine storm in asubject undergoing dendritic cell therapy, comprises the step ofadministering a composition comprising apoptotic cells or apoptotic cellsupernatants or a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment thereof or analogue thereof, a tellurium-based compound, or animmune modulating agent, or any combination thereof, to said subject. Inanother embodiment, a method of alleviating cytokine release syndrome orcytokine storm in a subject undergoing dendritic cell therapy, comprisesthe step of administering a composition comprising apoptotic cells orapoptotic cell supernatants or a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment thereof or analogue thereof, a tellurium-basedcompound, or an immune modulating agent, or any combination thereof, tosaid subject.

In one embodiment, disclosed herein are a composition comprisingdendritic cells and an additional agent, wherein said additional agentcomprises apoptotic cells, apoptotic supernatants, a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment thereof or analogue thereof,a tellurium-based compound, or an immune modulating agent, or anycombination thereof.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm comprising the step of administering acomposition comprising apoptotic cells or apoptotic cell supernatants ora CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment thereof oranalogue thereof, a tellurium-based compound, or an immune modulatingagent, or any combination thereof, to said subject.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of autoimmune toxicity, said method comprisingthe step of administering a composition comprising apoptotic cells or anapoptotic cell population, or an apoptotic cell supernatant orcomposition thereof or a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, a tellurium-based compound, oran immune modulating agent, or any combination thereof, to said subject.

Alpha-1-Antitrypsin (AAT)

Alpha-1-antitrypsin (AAT) is a circulating 52-kDa glycoprotein that isproduced mainly by the liver. AAT is primarily known as a serineprotease inhibitor and is encoded by the gene SERPINA1. AAT inhibitsneutrophil elastase, and inherited deficiency in circulating AAT resultsin lung-tissue deterioration and liver disease. Serum AAT concentrationsin healthy individuals increase twofold during inflammation.

There is a negative association between AAT levels and the severity ofseveral inflammatory diseases. For example, reduced levels or activityof AAT have been described in patients with HIV infection, diabetesmellitus, hepatitis C infection-induced chronic liver disease, andseveral types of vasculitis.

Increasing evidence demonstrates that human serum derivedalpha-1-anti-trypsin (AAT) reduces production of pro-inflammatorycytokines, induces anti-inflammatory cytokines, and interferes withmaturation of dendritic cells.

Indeed, the addition of AAT to human peripheral blood mononuclear cells(PBMC) inhibits LPS induced release of TNF-α and IL-1β but increasesIL-1 receptor antagonist (IL-1Ra) and IL-10 production.

AAT reduces in vitro IL-1β-mediated pancreatic islet toxicity, and AATmonotherapy prolongs islet allograft survival, promotes antigen-specificimmune tolerance in mice, and delays the development of diabetes innon-obese diabetic (NOD) mice. AAT was shown to inhibit LPS-inducedacute lung injury in experimental models. Recently, AAT was shown toreduce the size of infarct and the severity of heart failure in a mousemodel of acute myocardial ischemia-reperfusion injury.

Monotherapy with clinical-grade human AAT (hAAT) reduced circulatingpro-inflammatory cytokines, diminished Graft vs Host Disease (GvHD)severity, and prolonged animal survival after experimental allogeneicbone marrow transfer (Tawara et al., Proc Natl Acad Sci USA. 2012 Jan.10; 109(2):564-9), incorporated herein by reference. AAT treatmentreduced the expansion of alloreactive T effector cells but enhanced therecovery of T regulatory T-cells, (Tregs) thus altering the ratio ofdonor T effector to T regulatory cells in favor of reducing thepathological process. In vitro, AAT suppressed LPS-induced in vitrosecretion of proinflammatory cytokines such as TNF-α and IL-1β, enhancedthe production of the anti-inflammatory cytokine IL-10, and impairedNF-κB translocation in the host dendritic cells. Marcondes, Blood. 2014(Oct. 30; 124(18):2881-91) incorporated herein by reference show thattreatment with AAT not only ameliorated GvHD but also preserved andperhaps even enhanced the graft vs leukemia (GVL) effect.

In one embodiment, disclosed herein are compositions comprising chimericantigen receptor-expressing T-cells (CAR T-cells) andAlpha-1-antitrypsin (AAT). In another embodiment, CAR T-cells andAlpha-1-antitrypsin (AAT) are in separate compositions. In anotherembodiment, AAT comprises a full length AAT or a functional fragmentthereof. In another embodiment, AA comprises an analogue of a fulllength AAT or a functional fragment thereof. In another embodiment, acomposition comprising AAT further comprises apoptotic cells or anapoptotic cell supernatant.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) therapy, comprising the step of administering a compositioncomprising Alpha-1-antitrypsin (AAT) to said subject. In anotherembodiment, a method of treating cytokine release syndrome or a cytokinestorm in a subject undergoing chimeric antigen receptor-expressingT-cell (CAR T-cell) therapy, comprises the step of administering acomposition comprising Alpha-1-antitrypsin (AAT) to said subject. Inanother embodiment, a method of preventing cytokine release syndrome ora cytokine storm in a subject undergoing chimeric antigenreceptor-expressing T-cell (CAR T-cell) therapy, comprises the step ofadministering a composition comprising Alpha-1-antitrypsin (AAT) to saidsubject. In another embodiment, a method of ameliorating cytokinerelease syndrome or a cytokine storm in a subject undergoing chimericantigen receptor-expressing T-cell (CAR T-cell) therapy, comprises thestep of administering a composition comprising Alpha-1-antitrypsin (AAT)to said subject. In another embodiment, a method of alleviating cytokinerelease syndrome or a cytokine storm in a subject undergoing chimericantigen receptor-expressing T-cell (CAR T-cell) therapy, comprises thestep of administering a composition comprising Alpha-1-antitrypsin (AAT)to said subject.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm, comprising the step of administering acomposition comprising Alpha-1-antitrypsin (AAT) to said subject.

In one embodiment, AAT is administered alone to control cytokinerelease. In another embodiment, both AAT and apoptotic cells or acomposition thereof, or apoptotic cell supernatants or a compositionthereof, are administered to control cytokine release.

Immuno-Modulatory Agents

A skilled artisan would appreciate that immune-modulating agents mayencompass extracellular mediators, receptors, mediators of intracellularsignaling pathways, and regulators of translation and transcription. Inone embodiment, an additional agent disclosed herein is animmune-modulatory agent known in the art. In another embodiment, use inthe methods disclosed here of an immune-modulatory agent reduces thelevel of at least one cytokine. In another embodiment, use in themethods disclosed here of an immune-modulatory agent reduces or inhibitsCRS or a cytokine storm.

In one embodiment, an immune-modulatory agent comprises compounds thatblock, inhibit or reduce the release of cytokines or chemokines. Inanother embodiment, an immune-modulatory agent comprises compounds thatblock, inhibit or reduce the release of IL-21 or IL-23, or a combinationthereof. In another embodiment, an immune-modulatory agent comprises anantiretroviral drug in the chemokine receptor-5 (CCR5) receptorantagonist class, for example maraviroc. In another embodiment, animmune-modulatory agent comprises an anti-DNAM-1 antibody. In anotherembodiment, an immune-modulatory agent comprisesdamage/pathogen-associated molecules (DAMPs/PAMPs) selected from thegroup comprising heparin sulfate, ATP, and uric acid, or any combinationthereof. In another embodiment, an immune-modulatory agent comprises asialic acid binding Ig-like lectin (Siglecs). In another embodiment, animmune-modulatory agent comprises a cellular mediator of tolerance, forexample regulatory CD4⁺CD25⁺ T cells (Tregs) or invariant natural killerT cells (iNK T-cells). In another embodiment, an immune-modulatory agentcomprises JAK2 or JAK3 inhibitors selected from the group comprisingruxolitinib and tofacitinib. In another embodiment, an immune-modulatoryagent comprises an inhibitor of spleen tyrosine kinase (Syk), forexample fostamatinib. In another embodiment, an immune-modulatory agentcomprises histone deacetylase inhibitor vorinostat acetylated STAT3. Inanother embodiment, an immune-modulatory agent comprises neddylationinhibitors, for example MLN4924. In another embodiment, animmune-modulatory agent comprises an miR-142 antagonist. In anotherembodiment, an immune-modulatory agent comprises a chemical analogue ofcytidine, for example Azacitidine. In another embodiment, animmune-modulatory agent comprises an inhibitor of histone deacetylase,for example Vorinostat. In another embodiment, an immune-modulatoryagent comprises an inhibitor of histone methylation.

Tellurium-Based Compounds

Tellurium is a trace element found in the human body. Various telluriumcompounds, have immune-modulating properties, and have been shown tohave beneficial effects in diverse preclinical and clinical studies. Inone embodiment, methods disclosed herein comprise administration of atellurium-based compound as an additional agent.

In one embodiment, a tellurium-based compound inhibits the secretion ofat least one cytokine. In another embodiment, a tellurium-based compoundreduces the secretion of at least one cytokine. In another embodiment, atellurium-based compound inhibits or reduces a cytokine release syndrome(CRS) of a cytokine storm.

In one embodiment, disclosed herein are compositions comprising chimericantigen receptor-expressing T-cells (CAR T-cells) and a tellurium-basedcompound. In another embodiment, CAR T-cells and Tellurium-basedcompound are in separate compositions. In another embodiment, AATcomprises a full length AAT or a functional fragment thereof. In anotherembodiment, AA comprises an analogue of a full length AAT or afunctional fragment thereof

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) therapy, comprising the step of administering a compositioncomprising a Tellurium-based compound to said subject. In anotherembodiment, a method of treating cytokine release syndrome or a cytokinestorm in a subject undergoing chimeric antigen receptor-expressingT-cell (CAR T-cell) therapy, comprises the step of administering acomposition comprising a Tellurium-based compound to said subject. Inanother embodiment, a method of preventing cytokine release syndrome ora cytokine storm in a subject undergoing chimeric antigenreceptor-expressing T-cell (CAR T-cell) therapy, comprises the step ofadministering a composition comprising a Tellurium-based compound tosaid subject. In another embodiment, a method of ameliorating cytokinerelease syndrome or a cytokine storm in a subject undergoing chimericantigen receptor-expressing T-cell (CAR T-cell) therapy, comprises thestep of administering a composition comprising a Tellurium-basedcompound to said subject. In another embodiment, a method of alleviatingcytokine release syndrome or a cytokine storm in a subject undergoingchimeric antigen receptor-expressing T-cell (CAR T-cell) therapy,comprises the step of administering a composition comprising aTellurium-based compound to said subject.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm, comprising the step of administering acomposition comprising a Tellurium-based compound to said subject.

In one embodiment, a tellurium-based compound is administered alone tocontrol cytokine release. In another embodiment, both a tellurium-basedcompound and apoptotic cells or a composition thereof, or apoptotic cellsupernatants or a composition thereof, are administered to controlcytokine release.

Genetic Modification

In one embodiment, genetic modification of T-cells, dendritic cells,and/or apoptotic cells may be accomplished using RNA, DNA, recombinantviruses, or a combination thereof. In one embodiment, vectors derivedfrom gamma retroviruses or lentiviruses are used in the compositions andmethods as disclosed herein. In another embodiment, these vectors canintegrate into the host genome, with potentially permanent expression ofthe transgene and have low intrinsic immunogenicity. In anotherembodiment, another vector that integrates into the host genome and/orhas low intrinsic immunogenicity may be used in the compositions andmethods as disclosed herein. In another embodiment, thenon-viral-vector-mediated sleeping beauty transposon system is used toinsert the CAR and other genes into the T-cell. In another embodiment,“suicide genes” are integrated into the T-cells, in which expression ofa pro-apoptotic gene is under the control of an inducible promoterresponsive to a systemically delivered drug.

In one embodiment, genetic modification may be transient. In anotherembodiment, genetic modification may utilize messenger RNA (mRNA). Inanother embodiment, large numbers of cells may be infused on multipleoccasions in transiently engineered T-cells, such as mRNA-transfectedT-cells. In another embodiment, RNA-based electroporation of lymphocytesusing in vitro-transcribed mRNA mediates transient expression ofproteins for approximately one week and obviates the risk of integratingviral vectors. In another embodiment, mRNA-transduced dendritic cells ormRNA-electroporated T and NK lymphocytes.

It has been demonstrated that genetically modified T-cells can persistafter adoptive transfer for more than a decade without adverse effects,indicating that genetically modifying human T-cells is fundamentallysafe.

In another embodiment, the genetic modification of the compositions andin the methods as disclosed herein may be any method that is known inthe art.

Apoptotic Cells

In one embodiment, apoptotic cells (“Apocells”) for use in compositionsand methods as disclosed herein are as described in WO 2014/087408,which is incorporated by reference herein in its entirety. According tosome embodiments, the production method of the invention advantageouslyallows induction of an early-apoptosis state substantially withoutinduction of necrosis, wherein the cells remain stable at saidearly-apoptotic state for about 24 hours following preparation. Inanother embodiment, apoptotic cells for use in compositions and methodsas disclosed herein are produced in any way that is known in the art. Inanother embodiment, apoptotic cells for use in compositions and methodsdisclosed herein are autologous with a subject undergoing therapy. Inanother embodiment, apoptotic cells for use in compositions and methodsdisclosed herein are allogeneic with a subject undergoing therapy. Inanother embodiment, a composition comprising apoptotic cells comprisesapoptotic cells as disclosed herein or as is known in art.

In one embodiment, apoptotic cells comprise a cell preparationcomprising mononuclear-enriched cells, wherein the preparation comprisesat least 85% mononuclear cells, wherein at least 40% of the cells in thepreparation are in an early-apoptotic state, wherein at least 85% of thecells in the preparation are viable cells and wherein the preparationcomprises no more than 15% CD15^(high) expressing cells.

A skilled artisan would appreciate that the term “early-apoptotic state”may encompass cells that show early signs of apoptosis without latesigns of apoptosis. Examples of early signs of apoptosis in cellsinclude exposure of phosphatidylserine (PS) and the loss ofmitochondrial membrane potential. Examples of late events includepropidium iodide (PI) admission into the cell and the final DNA cutting.In order to document that cells are in an “early apoptotic” state, inone embodiment, PS exposure detection by Annexin-V and PI staining areused, and cells that are stained with Annexin V but not with PI areconsidered to be “early apoptotic cells”. In another embodiment, cellsthat are stained by both Annexin-V FITC and PI are considered to be“late apoptotic cells”. In another embodiment, cells that do not stainfor either Annexin-V or PI are considered non-apoptotic viable cells.

In one embodiment, apoptotic cells comprise cells in an early apoptoticstate. In another embodiment, apoptotic cells comprise cells wherein atleast 90% of said cells are in an early apoptotic state. In anotherembodiment, apoptotic cells comprise cells wherein at least 80% of saidcells are in an early apoptotic state. In another embodiment, apoptoticcells comprise cells wherein at least 70% of said cells are in an earlyapoptotic state. In another embodiment, apoptotic cells comprise cellswherein at least 60% of said cells are in an early apoptotic state. Inanother embodiment, apoptotic cells comprise cells wherein at least 50%of said cells are in an early apoptotic state.

In one embodiment, the composition comprising apoptotic cells furthercomprises an anti-coagulant.

In one embodiment, the anti-coagulant is selected from the groupconsisting of: heparin, acid citrate dextrose (ACD) Formula A and acombination thereof.

In one embodiment, the composition further comprises methylprednisolone.At one embodiment, the concentration of methylprednisolone does notexceed 30 μg/ml. In one embodiment, about 140×10⁶-210×10⁶ apoptoticcells are administered.

In one embodiment, the apoptotic cells are used at a high dose. In oneembodiment, the apoptotic cells are used at a high concentration. In oneembodiment, human apoptotic polymorphonuclear neutrophils (PMNs) areused. In one embodiment, a group of cells, of which 50% are apoptoticcells, are used. In one embodiment, apoptotic cells are verified byMay-Giemsa-stained cytopreps. In one embodiment, viability of cells areassessed by trypan blue exclusion. In one embodiment, the apoptotic andnecrotic status of the cells are confirmed by annexin V/propidium iodidestaining with detection by FACS.

In one embodiment, a dose of 10×10⁶ apoptotic cells is administered. Inanother embodiment, a dose of 10×10⁷ apoptotic cells is administered. Inanother embodiment, a dose of 10×10⁸ apoptotic cells is administered. Inanother embodiment, a dose of 10×10⁹ apoptotic cells is administered. Inanother embodiment, a dose of 10×10¹⁰ apoptotic cells is administered.In another embodiment, a dose of 10×10¹¹ apoptotic cells isadministered. In another embodiment, a dose of 10×10¹² apoptotic cellsis administered. In another embodiment, a dose of 10×10⁵ apoptotic cellsis administered. In another embodiment, a dose of 10×10⁴ apoptotic cellsis administered. In another embodiment, a dose of 10×10³ apoptotic cellsis administered. In another embodiment, a dose of 10×10² apoptotic cellsis administered.

In one embodiment, a high dose of apoptotic cells is administered. Inone embodiment, a dose of 35×10⁶ apoptotic cells is administered. Inanother embodiment, a dose of 210×10⁶ apoptotic cells is administered.In another embodiment, a dose of 70×10⁶ apoptotic cells is administered.In another embodiment, a dose of 140×10⁶ apoptotic cells isadministered. In another embodiment, a dose of 35-210×10⁶ apoptoticcells is administered.

According to some embodiments, obtaining a mononuclear-enriched cellcomposition according to the production method disclosed herein iseffected by leukapheresis. A skilled artisan would appreciate that theterm “leukapheresis” may encompass an apheresis procedure in whichleukocytes are separated from the blood of a donor. According to someembodiments, the blood of a donor undergoes leukapheresis and thus amononuclear-enriched cell composition is obtained according to theproduction method disclosed herein. It is to be noted, that the use ofat least one anticoagulant during leukapheresis is required, as is knownin the art, in order to prevent clotting of the collected cells.

According to some embodiments, the leukapheresis procedure is configuredto allow collection of mononuclear-enriched cell composition accordingto the production method disclosed herein. According to someembodiments, cell collections obtained by leukapheresis comprise atleast 65%. In other embodiments, at least 70%, or at least 80%mononuclear cells. Each possibility represents a separate embodiment asdisclosed herein. According to some embodiments, blood plasma from thecell-donor is collected in parallel to obtaining of themononuclear-enriched cell composition according to the production methoddisclosed herein. According to some embodiments, about 300-600 ml ofblood plasma from the cell-donor are collected in parallel to obtainingthe mononuclear-enriched cell composition according to the productionmethod disclosed herein. According to some embodiments, blood plasmacollected in parallel to obtaining the mononuclear-enriched cellcomposition according to the production method disclosed herein is usedas part of the freezing and/or incubation medium. Each possibilityrepresents a separate embodiment as disclosed herein. Additionaldetailed methods of obtaining an enriched population of apoptotic cellsfor use in the compositions and methods as disclosed herein may be foundin WO 2014/087408, which is incorporated herein by reference in itsentirety.

It is to be noted that, according to some embodiments, that while theinitial mononuclear-enriched cell preparation comprises at least 65%mononuclear cells, at least 70%, or at least 80% mononuclear cells, thefinal pharmaceutical composition disclosed herein, following theproduction method disclosed herein, comprises at least 85%. In anotherembodiment, at least 90%, or at least 95% mononuclear cells. Eachpossibility represents a separate embodiment as disclosed herein.

In one embodiment, the apoptotic cells may be administered by any methodknown in the art including, but not limited to, intravenous,subcutaneous, intranodal, intratumoral, intrathecal, intrapleural,intraperitoneal and directly to the thymus.

In one embodiment, the apoptotic cells are allogeneic. In one embodimentthe apoptotic cells are from pooled third party donors. In oneembodiment, the methods as disclosed herein comprise an additional stepthat is useful in overcoming rejection of allogeneic donor cells,including one or more steps described in U.S. Patent Application20130156794, which is incorporated herein by reference in its entirety.In one embodiment, the methods comprise the step of full or partiallymphodepletion prior to administration of the apoptotic cells, which inone embodiment, are allogeneic apoptotic cells. In one embodiment, thelymphodepletion is adjusted so that it delays the host versus graftreaction for a period sufficient to allow the allogeneic apoptotic cellsto control cytokine release. In another embodiment, the methods comprisethe step of administering agents that delay egression of the allogeneicapoptotic T-cells from lymph nodes, such as2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol (FTY720),5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)pheny-1]1,2,4-oxadiazole(SEW2871), 3-(2-(-hexylphenylamino)-2-oxoethylamino)propanoic acid(W123),2-ammonio-4-(2-chloro-4-(3-phenoxyphenylthio)phenyl)-2-(hydroxymethyl)but-ylhydrogen phosphate (KRP-203 phosphate) or other agents known in the art,may be used as part of the compositions and methods as disclosed hereinto allow the use of allogeneic apoptotic cells having efficacy andlacking initiation of graft vs host disease. In another embodiment, MHCexpression by the allogeneic apoptotic T-cells is silenced to reduce therejection of the allogeneic cells.

In another embodiment, the methods comprise the step of irradiatingapoptotic cells derived from WBCs from a donor prior to administrationto a recipient. In one embodiment, cells are irradiated in a way thatwill avoid proliferation and/or activation of residual viable cellswithin the apoptotic cell population. In another embodiment, theirradiated apoptotic cells preserve all their early apoptotic-, immunemodulation-, stability-properties. In another embodiment, theirradiation step uses UV radiation. In another embodiment, the radiationstep uses gamma radiation. In another embodiment, the apoptotic cellscomprise a decreased percent of living non-apoptotic cells, comprise apreparation having a suppressed cellular activation of any livingnon-apoptotic cells present within the apoptotic cell preparation, orcomprise a preparation having reduced proliferation of any livingnon-apoptotic cells present within the apoptotic cell preparation, orany combination thereof.

In one embodiment, a pooled mononuclear apoptotic cell preparationcomprising mononuclear cells in an early apoptotic state, wherein saidpooled mononuclear apoptotic cells comprise a decreased percent ofliving non-apoptotic cells, a preparation having a suppressed cellularactivation of any living non-apoptotic cells, or a preparation havingreduced proliferation of any living non-apoptotic cells, or anycombination thereof. In another embodiment, the pooled mononuclearapoptotic cells have been irradiated. In another embodiment, disclosedherein is a pooled mononuclear apoptotic cell preparation that in someembodiments, originates from the white blood cell fraction (WBC)obtained from donated blood.

In one embodiment, the apoptotic cell preparation is irradiated. Inanother embodiment, said irradiation comprises gamma irradiation or UVirradiation. In yet another embodiment, the irradiated preparation has areduced number of non-apoptotic cells compared with a non-irradiatedapoptotic cell preparation. In another embodiment, the irradiatedpreparation has a reduced number of proliferating cells compared with anon-irradiated apoptotic cell preparation. In another embodiment, theirradiated preparation has a reduced number of potentiallyimmunologically active cells compared with a non-irradiated apoptoticcell population.

In one embodiment, pooled blood comprises 3rd party blood not matchedbetween donor and recipient.

A skilled artisan would appreciate that the term “pooled” may encompassblood collected from multiple donors, prepared and possibly stored forlater use. This combined pool of blood may then be processed to producea pooled mononuclear apoptotic cell preparation. In another embodiment,a pooled mononuclear apoptotic cell preparation ensures that a readilyavailable supply of mononuclear apoptotic cells is available. In anotherembodiment, cells are pooled just prior to the incubation step whereinapoptosis is induced. In another embodiment, cells are pooled followingthe incubation step at the step of resuspension. In another embodiment,cells are pooled just prior to an irradiation step. In anotherembodiment, cells are pooled following an irradiation step. In anotherembodiment, cells are pooled at any step in the methods of preparation.

In one embodiment, a pooled apoptotic cell preparation is derived fromcells present in between about 2 and 25 units of blood. In anotherembodiment, said pooled apoptotic cell preparation is comprised of cellspresent in between about 2-5, 2-10, 2-15, 2-20, 5-10, 5-15, 5-20, 5-25,10-15, 10-20, 10-25, 6-13, or 6-25 units of blood. In anotherembodiment, said pooled apoptotic cell preparation is comprised of cellspresent in about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24 or 25 units of blood. The number of units ofblood needed is also dependent upon the efficiency of WBC recovery fromblood. For example, low efficiency WBC recovery would lead to the needfor additional units, while high efficiency WBC recovery would lead tofewer units needed. In some embodiments, each unit is a bag of blood. Inanother embodiment, a pooled apoptotic cell preparation is comprised ofcells present in at least 25 units of blood, at least 50 units of blood,or at least 100 units of blood. Each possibility represents a separateembodiment as disclosed herein.

In one embodiment, the units of blood comprise white blood cell (WBC)fractions from blood donations. In another embodiment, the donations maybe from a blood center or blood bank. In another embodiment, thedonations may be from donors in a hospital gathered at the time ofpreparation of the pooled apoptotic cell preparation. In anotherembodiment, units of blood comprising WBCs from multiple donors aresaved and maintained in an independent blood bank created for thepurpose of compositions and methods thereof as disclosed herein. Inanother embodiment, a blood bank developed for the purpose ofcompositions and methods thereof as disclosed herein, is able to supplyunits of blood comprising WBC from multiple donors and comprises aleukapheresis unit.

In one embodiment, the units of pooled WBCs are not restricted by HLAmatching. Therefore, the resultant pooled apoptotic cell preparationcomprises cell populations not restricted by HLA matching. Accordingly,in certain embodiments a pooled mononuclear apoptotic cell preparationcomprises allogeneic cells.

An advantage of a pooled mononuclear apoptotic cell preparation that isderived from pooled WBCs not restricted by HLA matching, is a readilyavailable source of WBCs and reduced costs of obtaining WBCs.

In one embodiment, pooled blood comprises blood from multiple donorsindependent of HLA matching. In another embodiment, pooled bloodcomprises blood from multiple donors wherein HLA matching with therecipient has been taken into consideration. For example, wherein 1 HLAallele, 2 HLA alleles, 3 HLA alleles, 4 HLA alleles, 5 HLA alleles, 6HLA alleles, or 7 HLA alleles have been matched between donors andrecipient. In another embodiment, multiple donors are partially matched,for example some of the donors have been HLA matched wherein 1 HLAallele, 2 HLA alleles, 3 HLA alleles, 4 HLA alleles, 5 HLA alleles, 6HLA alleles, or 7 HLA alleles have been matched between some of thedonors and recipient. Each possibility comprises an embodiment asdisclosed herein.

In certain embodiments, some viable non-apoptotic cells (apoptosisresistant) may remain following the induction of apoptosis stepdescribed below. The presence of these viable non-apoptotic cells is, inone embodiment, observed prior to an irradiation step. These viablenon-apoptotic cells may be able to proliferate or be activated. In oneembodiment, the pooled mononuclear apoptotic cell preparation derivedfrom multiple donors may be activated against the host, activatedagainst one another, or both.

In one embodiment, an irradiated cell preparation as disclosed hereinhas suppressed cellular activation and reduced proliferation comparedwith a non-irradiated cell preparation. In another embodiment, theirradiation comprises gamma irradiation or UV irradiation. In anotherembodiment, an irradiated cell preparation has a reduced number ofnon-apoptotic cells compared with a non-irradiated cell preparation. Inanother embodiment, the irradiation comprises about 15 Grey units (Gy).In another embodiment, the irradiation comprises about 20 Grey units(Gy). In another embodiment, the irradiation comprises about 25 Greyunits (Gy). In another embodiment, the irradiation comprises about 30Grey units (Gy). In another embodiment, the irradiation comprises about35 Grey units (Gy). In another embodiment, the irradiation comprisesabout 40 Grey units (Gy). In another embodiment, the irradiationcomprises about 45 Grey units (Gy). In another embodiment, theirradiation comprises about 50 Grey units (Gy). In another embodiment,the irradiation comprises about 55 Grey units (Gy). In anotherembodiment, the irradiation comprises about 60 Grey units (Gy). Inanother embodiment, the irradiation comprises about 65 Grey units (Gy).In another embodiment, the irradiation comprises up to 2500 Gy. Inanother embodiment, an irradiated pooled apoptotic cell preparationmaintains the same or a similar apoptotic profile, stability andefficacy as a non-irradiated pooled apoptotic cell preparation.

In one embodiment, a pooled mononuclear apoptotic cell preparation asdisclosed herein is stable for up to 24 hours. In another embodiment, apooled mononuclear apoptotic cell preparation is stable for at least 24hours. In another embodiment, a pooled mononuclear apoptotic cellpreparation is stable for more than 24 hours. In yet another embodiment,a pooled mononuclear apoptotic cell preparation as disclosed herein isstable for up to 36 hours. In still another embodiment, a pooledmononuclear apoptotic cell preparation is stable for at least 36 hours.In a further embodiment, a pooled mononuclear apoptotic cell preparationis stable for more than 36 hours. In another embodiment, a pooledmononuclear apoptotic cell preparation as disclosed herein is stable forup to 48 hours. In another embodiment, a pooled mononuclear apoptoticcell preparation is stable for at least 48 hours. In another embodiment,a pooled mononuclear apoptotic cell preparation is stable for more than48 hours.

In one embodiment, methods of producing the pooled cell preparationcomprising an irradiation step preserves the early apoptotic, immunemodulation, and stability properties observed in an apoptoticpreparation derived from a single match donor wherein the cellpreparation may not include an irradiation step. In another embodiment,a pooled mononuclear apoptotic cell preparation as disclosed herein doesnot elicit a graft versus host disease (GVHD) response.

Irradiation of the cell preparation is considered safe in the art.Irradiation procedures are currently performed on a routine basis todonated blood to prevent reactions to WBC.

In another embodiment, the percent of apoptotic cells in a pooledmononuclear apoptotic cell preparation as disclosed herein is close to100%, thereby reducing the fraction of living non-apoptotic cells in thecell preparation. In one embodiment, the percent of apoptotic cells isat least 40%. In another embodiment, the percent of apoptotic cells isat least 50%. In yet another embodiment, the percent of apoptotic cellsis at least 60%. In still another embodiment, the percent of apoptoticcells is at least 70%. In a further embodiment, the percent of apoptoticcells is at least 80%. In another embodiment, the percent of apoptoticcells is at least 90%. In yet another embodiment, the percent ofapoptotic cells is at least 99%. Accordingly, a cell preparationcomprising a reduced or non-existent fraction of living non-apoptoticcells may in one embodiment provide a pooled mononuclear apoptotic cellpreparation that does not elicit GVHD in a recipient. Each possibilityrepresents an embodiment as disclosed herein.

Alternatively, in another embodiment, the percentage of livingnon-apoptotic WBC is reduced by specifically removing the living cellpopulation, for example by targeted precipitation. In anotherembodiment, the percent of living non-apoptotic cells may be reducedusing magnetic beads that bind to phosphatidylserine. In anotherembodiment, the percent of living non-apoptotic cells may be reducedusing magnetic beads that bind a marker on the cell surface ofnon-apoptotic cells but not apoptotic cells. In another embodiment, theapoptotic cells may be selected for further preparation using magneticbeads that bind to a marker on the cell surface of apoptotic cells butnot non-apoptotic cells. In yet another embodiment, the percentage ofliving non-apoptotic WBC is reduced by the use of ultrasound.

In one embodiment the apoptotic cells are from pooled third partydonors.

In one embodiment, a pooled cell preparation comprises at least one celltype selected from the group consisting of: lymphocytes, monocytes andnatural killer cells. In another embodiment, a pooled cell preparationcomprises an enriched population of mononuclear cells. In oneembodiment, a pooled mononuclear is a mononuclear enriched cellpreparation comprises cell types selected from the group consisting of:lymphocytes, monocytes and natural killer cells. In another embodiment,the mononuclear enriched cell preparation comprises no more than 15%,alternatively no more than 10%, typically no more than 5%polymorphonuclear leukocytes, also known as granulocytes (i.e.,neutrophils, basophils and eosinophils). In another embodiment, a pooledmononuclear cell preparation is devoid of granulocytes. Each possibilityrepresents a separate embodiment as disclosed herein.

In another embodiment, the pooled mononuclear enriched cell preparationcomprises no more than 15%, alternatively no more than 10%, typically nomore than 5% CD15^(high) expressing cells. In one embodiment, a pooledapoptotic cell preparation comprises less than 15% CD15 high expressingcells. Each possibility represents a separate embodiment as disclosedherein.

In one embodiment, the pooled mononuclear enriched cell preparationdisclosed herein comprises at least 80% mononuclear cells, at least 85%mononuclear cells, alternatively at least 90% mononuclear cells, or atleast 95% mononuclear cells, wherein each possibility is a separateembodiment disclosed herein. According to some embodiments, the pooledmononuclear enriched cell preparation disclosed herein comprises atleast 85% mononuclear cells.

In another embodiment, any pooled cell preparation that has a finalpooled percent of mononuclear cells of at least 80% is considered apooled mononuclear enriched cell preparation as disclosed herein. Thus,pooling cell preparations having increased polymorphonuclear cells (PMN)with cell preparations having high mononuclear cells with a resultant“pool” of at least 80% mononuclear cells comprises a preparation asdisclosed herein. According to some embodiments, mononuclear cellscomprise lymphocytes and monocytes.

A skilled artisan would appreciate that the term “mononuclear cells” mayencompass leukocytes having a one lobed nucleus. In another embodiment,a pooled apoptotic cell preparation as disclosed herein comprises lessthan 5% polymorphonuclear leukocytes.

In one embodiment, the apoptotic cells are T-cells. In anotherembodiment, the apoptotic cells are derived from the same pooled thirdparty donor T-cells as the CAR T-cells. In another embodiment, theapoptotic cells are derived from the CAR T-cell population.

Surprisingly, the apoptotic cells reduce production of cytokinesassociated with the cytokine storm such as IL-6. In one embodiment, theapoptotic cells affect cytokine expression levels in macrophages andDCs, but do not affect cytokine expression levels in the T-cellsthemselves. It was therefore unexpected that apoptotic cells would beuseful in enhancing CAR T-cell therapy or dendritic cell therapy.

In another embodiment, the apoptotic cells trigger death of T-cells, butnot via changes in cytokine expression levels.

In another embodiment, apoptotic cells antagonize the priming ofmacrophages and dendritic cells to secrete cytokines that wouldotherwise amplify the cytokine storm. In another embodiment, apoptoticcells increase Tregs which suppress the inflammatory response and/orprevent excess release of cytokines.

In one embodiment, administration of apoptotic cells inhibits one ormore pro-inflammatory cytokines. In one embodiment, the pro-inflammatorycytokine comprises IL-1beta, IL-6, TNF-alpha, or IFN-gamma, or anycombination thereof. In another embodiment, administration of apoptoticcells promotes the secretion of one or more anti-inflammatory cytokines.In one embodiment, the anti-inflammatory cytokine comprises TGF-beta,IL10, or PGE2, or any combination thereof.

In another embodiment, administration of apoptotic cells inhibitsdendritic cell maturation following exposure to TLR ligands. In anotherembodiment, administration of apoptotic cells creates potentiallytolerogenic dendritic cells, which in one embodiment, are capable ofmigration, and in one embodiment, the migration is due to CCR7. Inanother embodiment, administration of apoptotic cells elicits varioussignaling events which in one embodiment is TAM receptor signaling(Tyro3, Ax1 and Mer) which in one embodiment, inhibits inflammation inantigen-presenting cells.

In one embodiment, Tyro-3, Ax1, and Mer constitute the TAM family ofreceptor tyrosine kinases (RTKs) characterized by a conserved sequencewithin the kinase domain and adhesion molecule-like extracellulardomains. In another embodiment, administration of apoptotic cellsactivates signaling through MerTK. In another embodiment, administrationof apoptotic cells activates the phosphatidylinositol 3-kinase(PI3K)/AKT pathway, which in one embodiment, negatively regulates NF-κB.In another embodiment, administration of apoptotic cells negativelyregulates the inflammasome which in one embodiment leads to inhibitionof pro-inflammatory cytokine secretion, DC maturation, or a combinationthereof. In another embodiment, administration of apoptotic cellsupregulates expression of anti-inflammatory genes such as Nr4a, Thbs1,or a combination thereof. In another embodiment, administration ofapoptotic cells induces a high level of AMP which in one embodiment, isaccumulated in a Pannexin1-dependent manner. In another embodiment,administration of apoptotic cells suppresses inflammation.

Apoptotic Cell Supernatants (ApoSup and ApoSup Mon)

In one embodiment, compositions for use in the methods and treatments asdisclosed herein include an apoptotic cell supernatant as disclosedherein.

In one embodiment, the apoptotic cell supernatant is obtained by amethod comprising the steps of a) providing apoptotic cells, b)culturing the apoptotic cells of step a), and c) separating thesupernatant from the cells.

In one embodiment, apoptotic cells for use making an apoptotic cellsupernatant as disclosed herein are autologous with a subject undergoingtherapy. In another embodiment, apoptotic cells for use in making anapoptotic cell supernatant disclosed herein are allogeneic with asubject undergoing therapy.

The “apoptotic cells” from which the apoptotic cell supernatant isobtained may be cells chosen from any cell type of a subject, or anycommercially available cell line, subjected to a method of inducingapoptosis known to the person skilled in the art. The method of inducingapoptosis may be hypoxia, ozone, heat, radiation, chemicals, osmoticpressure, pH shift, X-ray irradiation, gamma-ray irradiation, UVirradiation, serum deprivation, corticoids or combinations thereof, orany other method described herein or known in the art. In anotherembodiment, the method of inducing apoptosis produces apoptotic cells inan early apoptotic state.

In one embodiment, the apoptotic cells are leukocytes.

In an embodiment, said apoptotic leukocytes are derived from peripheralblood mononuclear cells (PBMC). In another embodiment, said leukocytesare from pooled third party donors. In another embodiment, saidleukocytes are allogeneic.

According to one embodiment, the apoptotic cells are provided byselecting non-adherent leukocytes and submitting them to apoptosisinduction, followed by a cell culture step in culture medium.“Leukocytes” used to make the apoptotic cell-phagocyte supernatant maybe derived from any lineage, or sub-lineage, of nucleated cells of theimmune system and/or hematopoietic system, including but not limited todendritic cells, macrophages, masT-cells, basophils, hematopoietic stemcells, bone marrow cells, natural killer cells, and the like. Theleukocytes may be derived or obtained in any of various suitable ways,from any of various suitable anatomical compartments, according to anyof various commonly practiced methods, depending on the application andpurpose, desired leukocyte lineage, etc. In one embodiment, the sourceleukocytes are primary leukocytes. In another embodiment, the sourceleukocytes are primary peripheral blood leukocytes.

Primary lymphocytes and monocytes may be conveniently derived fromperipheral blood. Peripheral blood leukocytes include 70-95 percentlymphocytes, and 5-25 percent monocytes.

Methods for obtaining specific types of source leukocytes from blood areroutinely practiced. Obtaining source lymphocytes and/or monocytes canbe achieved, for example, by harvesting blood in the presence of ananticoagulant, such as heparin or citrate. The harvested blood is thencentrifuged over a Ficoll cushion to isolate lymphocytes and monocytesat the gradient interface, and neutrophils and erythrocytes in thepellet.

Leukocytes may be separated from each other via standard immunomagneticselection or immunofluorescent flow cytometry techniques according totheir specific surface markers, or via centrifugal elutriation. Forexample, monocytes can be selected as the CD14+ fraction, T-lymphocytescan be selected as CD3+ fraction, B-lymphocytes can be selected as theCD19+ fraction, macrophages as the CD206+ fraction.

Lymphocytes and monocytes may be isolated from each other by subjectingthese cells to substrate-adherent conditions, such as by static culturein a tissue culture-treated culturing recipient, which results inselective adherence of the monocytes, but not of the lymphocytes, to thecell-adherent substrate.

Leukocytes may also be obtained from peripheral blood mononuclear cells(PBMCs), which may be isolated as described herein.

One of ordinary skill in the art will possess the necessary expertise tosuitably culture primary leukocytes so as to generate desired quantitiesof cultured source leukocytes as disclosed herein, and ample guidancefor practicing such culturing methods is available in the literature ofthe art.

One of ordinary skill in the art will further possess the necessaryexpertise to establish, purchase, or otherwise obtain suitableestablished leukocyte cell lines from which to derive the apoptoticleukocytes. Suitable leukocyte cell lines may be obtained fromcommercial suppliers, such as the American Tissue Type Collection(ATCC). It will be evident to the person skilled in the art that sourceleukocytes should not be obtained via a technique which willsignificantly interfere with their capacity to produce the apoptoticleukocytes.

In an embodiment, the apoptotic cells comprise a cell preparationcomprising mononuclear-enriched cells, wherein the preparation comprisesat least 85% mononuclear cells, wherein at least 40% of the cells in thepreparation are in an early-apoptotic state, wherein at least 85% of thecells in the preparation are viable cells and wherein the preparationcomprises no more than 15% CD15^(high) expressing cells.

In another embodiment, the apoptotic cells may be apoptotic lymphocytes.Apoptosis of lymphocytes, such as primary lymphocytes, may be induced bytreating the primary lymphocytes with serum deprivation, acorticosteroid, or irradiation. In another embodiment, inducingapoptosis of primary lymphocytes via treatment with a corticosteroid iseffected by treating the primary lymphocytes with dexamethasone. Inanother embodiment, with dexamethasone at a concentration of about 1micromolar. In another embodiment, inducing apoptosis of primarylymphocytes via irradiation is effected by treating the primarylymphocytes with gamma-irradiation. In another embodiment, with a dosageof about 66 rad. Such treatment results in the generation of apoptoticlymphocytes suitable for the co-culture step with phagocytes.

In a further embodiment, apoptotic cells may be apoptotic monocytes,such as primary monocytes. To generate apoptotic monocytes the monocytesare subjected to in vitro conditions of substrate/surface-adherenceunder conditions of serum deprivation. Such treatment results in thegeneration of non-pro-inflammatory apoptotic monocytes suitable for theco-culture step with phagocytes.

In other embodiments, the apoptotic cells may be any apoptotic cellsdescribed herein, including allogeneic apoptotic cells, third partyapoptotic cells, and pools of apoptotic cells.

In other embodiments, the apoptotic cell supernatant may be obtainedthrough the co-culture of apoptotic cells with other cells.

Thus, in one embodiment, the apoptotic cell supernatant is an apoptoticcell supernatant obtained by a method comprising the steps of a)providing apoptotic cells, b) providing other cells, c) optionallywashing the cells from step a) and b), d) co-culturing the cells of stepa) and b), and optionally e) separating the supernatant from the cells.

In one embodiment, the other cells co-cultured with the apoptotic cellsare white blood cells.

Thus, in one embodiment, the apoptotic cell supernatant is an apoptoticcell-white blood cell supernatant obtained by a method comprising thesteps of a) providing apoptotic cells, b) providing white blood cells,c) optionally washing the cells from step a) and b), d) co-culturing thecells of step a) and b), and optionally e) separating the supernatantfrom the cells.

In one embodiment, the white blood cells may be phagocytes, such asmacrophages, monocytes or dendritic cells.

In one embodiment, the white blood cells may be B cells, T-cells, ornatural killer (NK cells).

Thus, in one embodiment, compositions for use in the methods andtreatments as disclosed herein include apoptotic cell-phagocytesupernatants as described in WO 2014/106666, which is incorporated byreference herein in its entirety. In another embodiment, apoptoticcell-phagocyte supernatants for use in compositions and methods asdisclosed herein are produced in any way that is known in the art.

In one embodiment, the apoptotic cell-phagocyte supernatant is obtainedfrom a co-culture of phagocytes with apoptotic cells,

In one embodiment, the apoptotic cell-phagocyte supernatant is obtainedby a method comprising the steps of a) providing phagocytes, b)providing apoptotic cells, c) optionally washing the cells from step a)and b), d) co-culturing the cells of step a) and b), and optionally e)separating the supernatant from the cells.

The term “phagocytes” denotes cells that protect the body by ingesting(phagocytosing) harmful foreign particles, bacteria, and dead or dyingcells. Phagocytes include for example cells called neutrophils,monocytes, macrophages, dendritic cells, and mast T-cells,preferentially dendritic cells and monocytes/macrophages. The phagocytesmay be dendritic cells (CD4+ HLA-DR+ Lineage-BDCA1/BDCA3+), macrophages(CD14+CD206+ HLA-DR+), or derived from monocytes (CD14+). Techniques todistinguish these different phagocytes are known to the person skilledin the art.

In an embodiment, monocytes are obtained by a plastic adherence step.Said monocytes can be distinguished from B and T-cells with the markerCD14+, whereas unwanted B cells express CD19+ and T-cells CD3+. AfterMacrophage Colony Stimulating Factor (M-CSF) induced maturation theobtained macrophages are in one embodiment, positive for the markersCD14+, CD206+, HLA-DR+.

In an embodiment, said phagocytes are derived from peripheral bloodmononuclear cells (PBMC).

Phagocytes may be provided by any method known in the art for obtainingphagocytes. In one embodiment, phagocytes such as macrophages ordendritic cells can be directly isolated from a subject or be derivedfrom precursor cells by a maturation step.

In one embodiment, macrophages may be directly isolated from theperitoneum cavity of a subject and cultured in complete RRPMI medium.Macrophages can also be isolated from the spleen.

Phagocytes are also obtainable from peripheral blood monocytes. In saidexample, monocytes when cultured differentiate into monocyte-derivedmacrophages upon addition of, without limitation to, macrophage colonystimulating factor (M-CSF) to the cell culture media.

For example, phagocytes may be derived from peripheral blood mononuclearcells (PBMC). For example, PBMC may be isolated from cytapheresis bagfrom an individual through Ficoll gradient centrifugation, plated in acell-adherence step for 90 min in complete RPMI culture medium (10% FBS,1% Penicillin/Streptomycin). Non-adherent T-cells are removed by aplastic adherence step, and adherent T-cells cultured in complete RPMImilieu supplemented with recombinant human M-CSF. After the cultureperiod, monocyte-derived macrophages are obtained.

Phagocytes can be selected by a cell-adherence step. Said “celladherence step” means that phagocytes or cells which can mature intophagocytes are selected via culturing conditions allowing the adhesionof the cultured cells to a surface, a cell adherent surface (e.g. atissue culture dish, a matrix, a sac or bag with the appropriate type ofnylon or plastic). A skilled artisan would appreciate that the term“Cell adherent surfaces” may encompass hydrophilic and negativelycharged, and may be obtained in any of various ways known in the art, Inanother embodiment by modifying a polystyrene surface using, forexample, corona discharge, or gas-plasma. These processes generatehighly energetic oxygen ions which graft onto the surface polystyrenechains so that the surface becomes hydrophilic and negatively charged.Culture recipients designed for facilitating cell-adherence thereto areavailable from various commercial suppliers (e.g. Corning, Perkin-Elmer,Fisher Scientific, Evergreen Scientific, Nunc, etc.).

B cells, T-cells and NK cells may be provided by any method known in theart for obtaining such cells. In one embodiment, B cells, T-cells or NKcells can be directly isolated from a subject or be derived fromprecursor cells by a maturation step. In another embodiment, the B, T orNK cells can be from a B, T or NK cell line. One of ordinary skill inthe art will possess the necessary expertise to establish, purchase, orotherwise obtain suitable established B cells, T-cells and NK celllines. Suitable cell lines may be obtained from commercial suppliers,such as the American Tissue Type Collection (ATCC).

In an embodiment, said apoptotic cells and said white blood cells, suchas the phagocytes, B, T or NK cells, are cultured individually prior tothe co-culture step d).

The cell maturation of phagocytes takes place during cell culture, forexample due to addition of maturation factors to the media. In oneembodiment said maturation factor is M-CSF, which may be used forexample to obtain monocyte-derived macrophages.

The culture step used for maturation or selection of phagocytes mighttake several hours to several days. In another embodiment saidpre-mature phagocytes are cultured for 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58 hours in an appropriate culture medium.

The culture medium for phagocytes is known to the person skilled in theart and can be for example, without limitation, RPMI, DMEM, X-vivo andUltraculture milieus.

In an embodiment, co-culture of apoptotic cells and phagocytes takesplace in a physiological solution.

Prior to this “co-culture”, the cells may be submitted to a washingstep. In one embodiment, the white blood cells (e.g. the phagocytes) andthe apoptotic cells are washed before the co-culture step. In anotherembodiment, the cells are washed with PBS.

During said co-culture the white blood cells (e.g. the phagocytes suchas macrophages, monocytes, or phagocytes, or the B, T or NK cells) andthe apoptotic cells may be mixed in a ratio of 10:1, 9:1; 8:1, 7:1, 6:1,5:1, 4:1, 3:1, 2:1, or 1:1, or in a ratio of (white bloodcells:apoptotic cells) 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.In one example, the ratio of white blood cells to apoptotic cells is1:5.

The co-culture of the cells might be for several hours to several days.In some embodiments, said apoptotic cells are cultured for 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52 hours. A person skilled in the art can evaluate theoptimal time for co-culture by measuring the presence ofanti-inflammatory compounds, the viable amount of white blood cells andthe amount of apoptotic cells which have not been eliminated so far.

The elimination of apoptotic cells by phagocytes is observable withlight microscopy due to the disappearance of apoptotic cells.

In one embodiment, the culture of apoptotic cells, such as theco-culture with culture with white blood cells (e.g. phagocytes such asmacrophages, monocytes, or phagocytes, or the B, T or NK cells), takesplace in culture medium and/or in a physiological solution compatiblewith administration e.g. injection to a subject.

A skilled artisan would appreciate that a “physiological solution” mayencompass a solution which does not lead to the death of white bloodcells within the culture time. In some embodiments, the physiologicalsolution does not lead to death over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 hours. Inother embodiment, 48 hours, or 30 hours.

In one embodiment, the white blood cells (e.g. phagocytes such asmacrophages, monocytes, or phagocytes, or the B, T or NK cells) and theapoptotic cells are incubated in the physiological solution for at least30 min. This time of culture allows phagocytosis initiation andsecretion of cytokines and other beneficial substances.

In an embodiment, such a physiological solution does not inhibitapoptotic leukocyte elimination by leukocyte-derived macrophages.

At the end of the culture or the co-culture step, the supernatant isoptionally separated from the cultured apoptotic cells or theco-cultured cells. Techniques to separate the supernatant from the cellsare known in the art. For example, the supernatant can be collectedand/or filtered and/or centrifuged to eliminate cells and debris. Forexample, said supernatant may be centrifuged at 3000 rpm for 15 minutesat room temperature to separate it from the cells.

The supernatant may be “inactivated” prior to use, for example byirradiation. Therefore, the method for preparing the apoptotic cellsupernatant may comprise an optional additional irradiation step f).Said “irradiation” step can be considered as a disinfection method thatuses X-ray irradiation (25-45 Gy) at sufficiently rate to killmicroorganisms, as routinely performed to inactivate blood products.

Irradiation of the supernatant is considered safe in the art.Irradiation procedures are currently performed on a routine basis todonated blood to prevent reactions to WBC.

In an embodiment, the apoptotic cell supernatant is formulated into apharmaceutical composition suitable for administration to a subject, asdescribed in detail herein.

In one embodiment, the final product is stored at +4° C. In anotherembodiment, the final product is for use in the next 48 hours.

In one embodiment, the apoptotic cell supernatant, such as an apoptoticcell-phagocyte supernatant, or pharmaceutical composition comprising thesupernatant, may be lyophilized, for example for storage at −80° C.

In one specific embodiment, as described in Example 1 of WO 2014/106666,an apoptotic cell-phagocyte supernatant may be made using thymic cellsas apoptotic cells. After isolation, thymic cells are irradiated (e.g.with a 35 X-Gray irradiation) and cultured in complete DMEM culturemedium for, for example, 6 hours to allow apoptosis to occur. Inparallel, macrophages are isolated from the peritoneum cavity, washedand cultured in complete RPMI (10% FBS, Peni-Strepto, EAA, Hepes, NaPand 2-MercaptoEthanol). Macrophages and apoptotic cells are then washedand co-cultured for another 48 hour period in phenol-free X-vivo mediumat a ⅕ macrophage/apoptotic cell ratio. Then, supernatant is collected,centrifuged to eliminate debris and may be frozen or lyophilized forconservation. Macrophage enrichment may be confirmed using positivestaining for F4/80 by FACS. Apoptosis may be confirmed by FACS usingpositive staining for Annexin-V and 7AAD exclusion.

In an embodiment, the apoptotic cell supernatant is enriched in TGF-βlevels both in active and latent forms of TGF-β, compared tosupernatants obtained from either macrophages or apoptotic cellscultured separately. In an embodiment, IL-10 levels are also increasedcompared to macrophages cultured alone and dramatically increasedcompared to apoptotic cells cultured alone. In another embodiment,inflammatory cytokines such as IL-6 are not detectable and IL-1β and TNFare undetectable or at very low levels.

In an embodiment, the apoptotic cell supernatant, when compared tosupernatants from macrophages cultured alone or from apoptotic cellscultured alone, has increased levels of IL-1ra, TIMP-1, CXCL1/KC andCCL2/JE/MCP1, which might be implicated in a tolerogenic role of thesupernatant to control inflammation, in addition to TGF-β and IL-10.

In another specific embodiment, as described in Example 3 of WO2014/106666, human apoptotic cell-phagocyte supernatant may be made fromthe co-culture of macrophages derived from peripheral blood mononuclearcells (PBMC) cultured with apoptotic PBMC. Thus, PBMC are isolated fromcytapheresis bag from a healthy volunteer through, for example, Ficollgradient centrifugation. Then PBMC are plated for 90 min in completeRPMI culture medium (10% FBS, 1% Penicillin/Streptomycin). Then,non-adherenT-cells are removed and rendered apoptotic using, forexample, a 35 Gy dose of X-ray irradiation and cultured in complete RPMImilieu for 4 days (including cell wash after the first 48 hrs ofculture), in order to allow apoptosis to occur. In parallel, adherentT-cells are cultured in complete RPMI milieu supplemented with 50 μg/mLof recombinant human M-CSF for 4 days including cell wash after thefirst 48 hrs. At the end of the 4-day culture period, monocyte-derivedmacrophages and apoptotic cells are washed and cultured together inX-vivo medium for again 48 hours at a one macrophage to 5 apoptotic cellratio. Then supernatant from the latter culture is collected,centrifuged to eliminate cells and debris, and may be frozen orlyophilized for conservation and subsequent use.

In an embodiment, as described in WO 2014/106666, human apoptoticcell-phagocyte supernatant may be obtained in 6 days from peripheralblood mononuclear cells (PBMC). Four days to obtain PBMC-derivedmacrophages using M-CSF addition in the culture, and 2 more days for theco-culture of PBMC-derived macrophages with apoptotic cells,corresponding to the non-adherent PBMC isolated at day 0.

In an embodiment, as described in WO 2014/106666, a standardized humanapoptotic cell-phagocyte supernatant may be obtained independently ofthe donor or the source of PBMC (cytapheresis or buffy coat). Theplastic-adherence step is sufficient to obtain a significant startingpopulation of enriched monocytes (20 to 93% of CD14+ cells afteradherence on plastic culture dish). In addition, such adherent T-cellsdemonstrate a very low presence of B and T-cells (1.0% of CD19+ B cellsand 12.8% of CD3+ T-cells). After 4 days of culture of adherent T-cellsin the presence of M-CSF, the proportion of monocytesderived-macrophages is significantly increased from 0.1% to 77.7% ofCD14+CD206+ HLA-DR+ macrophages. At that time, monocyte-derivedmacrophages may be co-cultured with apoptotic non-adherent PBMC (47.6%apoptotic as shown by annexin V staining and 7AAD exclusion) to producethe apoptotic cell-phagocyte supernatant during 48 hours.

In an embodiment, the collected apoptotic cell-phagocyte supernatant,contains significantly more latent TGF than in the culture supernatantof monocyte-derived macrophages alone or monocyte-derived macrophagestreated in inflammatory conditions (+LPS), and only contains trace orlow level of inflammatory cytokines such as IL-1β or TNF.

In one embodiment, the composition comprising the apoptotic cellsupernatant further comprises an anti-coagulant. In one embodiment, theanti-coagulant is selected from the group consisting of: heparin, acidcitrate dextrose (ACD) Formula A and a combination thereof.

In one embodiment, the composition comprising the apoptotic cellsupernatant further comprises methylprednisolone. At one embodiment, theconcentration of methylprednisolone does not exceed 30 μg/ml.

In one embodiment, the composition may be used at a total dose oraliquot of apoptotic cell supernatant derived from the co-culture ofabout 14×10⁹ of CD45+ cells obtained by cytapheresis equivalent to about200 million of cells per kilogram of body weight (for a 70 kg subject).In an embodiment, such a total dose is administered as unit doses ofsupernatant derived from about 100 million cells per kilogram bodyweight, and/or is administered as unit doses at weekly intervals, Inanother embodiment both of which. Suitable total doses according to thisembodiment include total doses of supernatant derived from about 10million to about 4 billion cells per kilogram body weight. In anotherembodiment, the supernatant is derived from about 40 million to about 1billion cells per kilogram body weight. In yet another embodiment thesupernatant is derived from about 80 million to about 500 million cellsper kilogram body weight. In still another embodiment, the supernatantis derived from about 160 million to about 250 million cells perkilogram body weight. Suitable unit doses according to this embodimentinclude unit doses of supernatant derived from about 4 million to about400 million cells per kilogram body weight. In another embodiment, thesupernatant is derived from about 8 million to about 200 million cellsper kilogram body weight. In another embodiment, the supernatant isderived from about 16 million to about 100 million cells per kilogrambody weight. In yet another embodiment, the supernatant is derived fromabout 32 million to about 50 million cells per kilogram body weight.

In another embodiment, a dose of apoptotic cell supernatant derived fromthe co-culture of about 10×10⁶ apoptotic cells is administered. Inanother embodiment, a dose derived from 10×10⁷ apoptotic cells isadministered. In another embodiment, a dose derived from 10×10⁸apoptotic cells is administered. In another embodiment, a dose derivedfrom 10×10⁹ apoptotic cells is administered. In another embodiment, adose derived from 10×10¹⁰ apoptotic cells is administered. In anotherembodiment, a dose derived from 10×10¹¹ apoptotic cells is administered.In another embodiment, a dose derived from 10×10¹² apoptotic cells isadministered. In another embodiment, a dose derived from 10×10⁵apoptotic cells is administered. In another embodiment, a dose derivedfrom 10×10⁴ apoptotic cells is administered. In another embodiment, adose derived from 10×10³ apoptotic cells is administered. In anotherembodiment, a dose derived from 10×10² apoptotic cells is administered.

In one embodiment, a dose of apoptotic cell supernatant derived from35×10⁶ apoptotic cells is administered. In another embodiment, a dosederived from 210×10⁶ apoptotic cells is administered. In anotherembodiment, a dose derived from 70×10⁶ apoptotic cells is administered.In another embodiment, a dose derived from 140×10⁶ apoptotic cells isadministered. In another embodiment, a dose derived from 35-210×10⁶apoptotic cells is administered.

In one embodiment, the apoptotic cell supernatant, or compositioncomprising said apoptotic cell supernatant, may be administered by anymethod known in the art including, but not limited to, intravenous,subcutaneous, intranodal, intratumoral, intrathecal, intrapleural,intraperitoneal and directly to the thymus, as discussed in detailherein.

Surprisingly, the apoptotic cell supernatants, such as apoptoticcell-phagocyte supernatants, reduces production of cytokines associatedwith the cytokine storm such as IL-6. Another cytokine, IL-2, is notinvolved in cytokine release syndrome although is secreted by DCs andmacrophages in small quantities. It is, however, required for thesurvival and proliferation of CAR-T-cells and is mostly produced bythese T-cells. Unexpectedly, the apoptotic cell supernatants, such asapoptotic cell-phagocyte supernatants, do not reduce IL-2 levelssufficiently to negatively affect the survival of CAR T-cells.

In one embodiment, the apoptotic cell supernatants, such as apoptoticcell-phagocyte supernatants, affect cytokine expression levels inmacrophages and DCs, but do not affect cytokine expression levels in theT-cells themselves. It was therefore unexpected that apoptotic cellsupernatants would be useful in enhancing CAR T-cell therapy ordendritic cell therapy.

In another embodiment, the apoptotic cell supernatants trigger death ofT-cells, but not via changes in cytokine expression levels.

In another embodiment, apoptotic cell supernatants, such as apoptoticcell-phagocyte supernatants antagonize the priming of macrophages anddendritic cells to secrete cytokines that would otherwise amplify thecytokine storm. In another embodiment, apoptotic cell supernatantsincrease Tregs which suppress the inflammatory response and/or preventexcess release of cytokines.

In one embodiment, administration of apoptotic cell supernatants, suchas apoptotic cell-phagocyte supernatants, inhibits one or morepro-inflammatory cytokines. In one embodiment, the pro-inflammatorycytokine comprises IL-1beta, IL-6, TNF-alpha, or IFN-gamma, or anycombination thereof. In another embodiment, administration of apoptoticcell supernatants promotes the secretion of one or moreanti-inflammatory cytokines. In one embodiment, the anti-inflammatorycytokine comprises TGF-beta, IL10, or PGE2, or any combination thereof.

In another embodiment, administration of apoptotic cell supernatants,such as apoptotic cell-phagocyte supernatants, inhibits dendritic cellmaturation following exposure to TLR ligands. In another embodiment,administration of apoptotic cell supernatants creates potentiallytolerogenic dendritic cells, which in one embodiment, are capable ofmigration, and in one embodiment, the migration is due to CCR7. Inanother embodiment, administration of apoptotic cell supernatantselicits various signaling events which in one embodiment is TAM receptorsignaling (Tyro3, Ax1 and Mer) which in one embodiment, inhibitsinflammation in antigen-presenting cells. In one embodiment, Tyro-3,Ax1, and Mer constitute the TAM family of receptor tyrosine kinases(RTKs) characterized by a conserved sequence within the kinase domainand adhesion molecule-like extracellular domains. In another embodiment,administration of apoptotic cell supernatants activates signalingthrough MerTK. In another embodiment, administration of apoptotic cellsupernatants activates the phosphatidylinositol 3-kinase (PI3K)/AKTpathway, which in one embodiment, negatively regulates NF-κB. In anotherembodiment, administration of apoptotic cell supernatants negativelyregulates the inflammasome which in one embodiment leads to inhibitionof pro-inflammatory cytokine secretion, DC maturation, or a combinationthereof. In another embodiment, administration of apoptotic cellsupernatants upregulates expression of anti-inflammatory genes such asNr4a, Thbs1, or a combination thereof. In another embodiment,administration of apoptotic cell supernatants induces a high level ofAMP which in one embodiment, is accumulated in a Pannexin1-dependentmanner. In another embodiment, administration of apoptotic cellsupernatants suppresses inflammation.

Compositions

In one embodiment, disclosed herein is a pharmaceutical composition forthe treatment of a condition or disease as described herein. In anotherembodiment, a pharmaceutical composition comprises a geneticallymodified immune cell or a genetically modified receptor thereof. Inanother embodiment, a genetically modified immune cell comprises aT-cell. In another embodiment, a genetically modified immune cellcomprises a chimeric antigen receptor CAR T-cell. In another embodiment,a genetically modified immune cell comprises a cytotoxic T lymphocyte.In another embodiment, a genetically modified immune cell comprises adendritic cell. In another embodiment, a genetically modified immunecell comprises a natural killer cell. In another embodiment, agenetically modified receptor comprises a T-cell receptor.

In still another embodiment, a pharmaceutical composition for thetreatment of a condition or a disease as described herein comprises aneffective amount of a genetically modified immune cell or a geneticallymodified receptor thereof, as described herein in a pharmaceuticallyacceptable excipient. In another embodiment, a pharmaceuticalcomposition for the treatment of a condition or a disease as describedherein comprises an effective amount of a CAR T-cell as described hereinin, and a pharmaceutically acceptable excipient. In another embodiment,a pharmaceutical composition for the treatment of a condition or adisease as described herein comprises an effective amount of a cytotoxicT cell, as described herein, and a pharmaceutically acceptableexcipient. In another embodiment, a pharmaceutical composition for thetreatment of a condition or a disease as described herein comprises aneffective amount of a genetically modified dendritic cell, as describedherein, and a pharmaceutically acceptable excipient. In anotherembodiment, a pharmaceutical composition for the treatment of acondition or a disease as described herein comprises an effective amountof a genetically modified natural killer cell, as described herein, anda pharmaceutically acceptable excipient. In another embodiment, apharmaceutical composition for the treatment of a condition or a diseaseas described herein comprises an effective amount of a geneticallymodified T-cell receptor, as described herein, and a pharmaceuticallyacceptable excipient.

In another embodiment, the condition or disease as described herein is atumor or cancer. In another embodiment, disclosed herein are acomposition comprising the genetically modified immune cell or receptorthereof, for example a CAR T-cell, that binds to a protein or peptide ofinterest as described herein. In another embodiment, the protein orpeptide of interest comprises a tumor antigen or a fragment thereof.

In another embodiment, a composition disclosed herein and used inmethods disclosed herein comprises apoptotic cells or an apoptotic cellsupernatant, and a pharmaceutically acceptable excipient. In yet anotherembodiment, a composition comprising an effective amount of agenetically modified immune cell or a genetically modified receptorthereof may be the same composition as comprises an apoptotic cellpopulation or an apoptotic cell supernatant. In another embodiment, acomposition comprising an effective amount of a CAR T-cell, or acytotoxic T-cell, or a genetically modified dendritic cell, or agenetically modified natural killer cell may be the same composition ascomprises an apoptotic cell population or an apoptotic cell supernatant.In yet another embodiment, a composition comprising an effective amountof genetically modified T-cell receptor may be the same composition ascomprises an apoptotic cell population or an apoptotic cell supernatant.In still another embodiment, a composition comprising an effectiveamount of a genetically modified immune cell selected from the groupcomprising a CAR T-cell, a cytotoxic T-cell, a natural killer cell, or adendritic cell, is not the same composition as comprises an apoptoticcell population or an apoptotic cell supernatant. In another embodiment,a composition comprises a chimeric antigen receptor-expressing T-cell(CAR T-cell) and either apoptotic cells or an apoptotic cellsupernatant, and a pharmaceutically acceptable excipient. In anotherembodiment, a composition comprising an effective amount of agenetically modified T-cell receptor is not the same composition ascomprises an apoptotic cell population or an apoptotic cell supernatant.

In another embodiment, apoptotic cells comprised in a compositioncomprise apoptotic cells in an early apoptotic state. In anotherembodiment, apoptotic cells comprised in a composition are pooled thirdparty donor cells. In another embodiment, an apoptotic cell supernatantcomprised in a composition disclosed herein is collected from earlyapoptotic cells. In another embodiment, an apoptotic cell supernatantcomprised in a composition disclosed herein, is collected pooled thirdparty donor cells.

In one embodiment, a composition comprising a genetically modifiedimmune cells, for example a CAR T-cell, further comprises an additionalpharmaceutical composition for preventing, suppressing, or modulatingcytokine release in a patient with cytokine release syndrome orexperiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a CART-cell, and apoptotic cells further comprises an additionalpharmaceutical composition for preventing, suppressing, or modulatingcytokine release in a patient with cytokine release syndrome orexperiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a CART-cell, and an apoptotic cell supernatant, further comprises anadditional pharmaceutical composition for preventing, suppressing, ormodulating cytokine release in a patient with cytokine release syndromeor experiencing a cytokine storm.

In one embodiment, a composition comprising a genetically modifiedimmune cells, for example a TCR T-cell, further comprises an additionalpharmaceutical composition for preventing, suppressing, or modulatingcytokine release in a patient with cytokine release syndrome orexperiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a TCRT-cell, and apoptotic cells further comprises an additionalpharmaceutical composition for preventing, suppressing, or modulatingcytokine release in a patient with cytokine release syndrome orexperiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a TCRT-cell, and an apoptotic cell supernatant, further comprises anadditional pharmaceutical composition for preventing, suppressing, ormodulating cytokine release in a patient with cytokine release syndromeor experiencing a cytokine storm.

In one embodiment, a composition comprising a genetically modifiedimmune cells, for example a dendritic cell, further comprises anadditional pharmaceutical composition for preventing, suppressing, ormodulating cytokine release in a patient with cytokine release syndromeor experiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a dendritic,and apoptotic cells further comprises an additional pharmaceuticalcomposition for preventing, suppressing, or modulating cytokine releasein a patient with cytokine release syndrome or experiencing a cytokinestorm. In another embodiment, a composition comprising a geneticallymodified immune cells, for example a dendritic, and an apoptotic cellsupernatant, further comprises an additional pharmaceutical compositionfor preventing, suppressing, or modulating cytokine release in a patientwith cytokine release syndrome or experiencing a cytokine storm.

In one embodiment, a composition comprising a genetically modifiedimmune cells, for example a NK cell, further comprises an additionalpharmaceutical composition for preventing, suppressing, or modulatingcytokine release in a patient with cytokine release syndrome orexperiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a NK cell,and apoptotic cells further comprises an additional pharmaceuticalcomposition for preventing, suppressing, or modulating cytokine releasein a patient with cytokine release syndrome or experiencing a cytokinestorm. In another embodiment, a composition comprising a geneticallymodified immune cells, for example a NK cell, and an apoptotic cellsupernatant, further comprises an additional pharmaceutical compositionfor preventing, suppressing, or modulating cytokine release in a patientwith cytokine release syndrome or experiencing a cytokine storm.

In one embodiment, the additional pharmaceutical composition comprises aCTLA-4 blocking agent, which in one embodiment is Ipilimumab. In anotherembodiment, the additional pharmaceutical composition comprises aalpha-1 anti-trypsin, as disclosed herein, or a fragment thereof, or ananalogue thereof. In another embodiment, the additional pharmaceuticalcomposition comprises a tellurium-based compound, a disclosed herein. Inanother embodiment, the additional pharmaceutical composition comprisesan immune modulating drug, as disclosed herein. In another embodiment,the additional pharmaceutical composition comprises a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment thereof or analogue thereof,a tellurium-based compound, or an immune modulating compound, or anycombination thereof.

In one embodiment, the composition comprising the genetically modifiedimmune cell, for example a CAR T-cell and the pharmaceutical compositioncomprising any one of a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, apoptotic cells, or anapoptotic cell supernatant, a tellurium-based compound, or an immunemodulating agent comprises a single composition. In another embodiment,the composition comprising the genetically modified immune cell, forexample CAR T-cells and the pharmaceutical composition comprising anyone of a CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragmentthereof or analogue thereof, apoptotic cells, or an apoptotic cellsupernatant, a tellurium-based compound, or an immune modulating agent,or any combination thereof, comprises multiple compositions, whereineach of the genetically modified immune cell, which in one embodiment isCAR T-cells, the CTLA-4 blocking agent, the alpha-1 anti-trypsin orfragment thereof or analogue thereof, the apoptotic cells, the apoptoticcell supernatant, the tellurium-based compound, or the immune modulatingagent, or any combination thereof, are comprised in a separatecomposition. In yet another embodiment, the composition comprising thegenetically modified immune cell, which in one embodiment is CAR T-cellsand the pharmaceutical composition comprising any one of a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, apoptotic cells, an apoptotic cell supernatant, atellurium-based compound, or an immune modulating agent, or anycombination thereof, comprises multiple compositions, wherein thegenetically modified immune cells, which in one embodiment are CART-cells, the CTLA-4 blocking agent, or the alpha-1 anti-trypsin orfragment thereof or analogue thereof, the tellurium-based compound, orthe immune modulating agent, or any combination thereof, or anycombination thereof are present in the genetically modified immune cell,for example a CAR T-cell, composition, and the apoptotic cells, or theapoptotic cell supernatant, are comprised in a separate composition.

In one embodiment, the composition comprising the genetically modifiedimmune cell, for example a TCR T-cell and the pharmaceutical compositioncomprising any one of a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, apoptotic cells, or anapoptotic cell supernatant, a tellurium-based compound, or an immunemodulating agent comprises a single composition. In another embodiment,the composition comprising the genetically modified immune cell, forexample TCR T-cells and the pharmaceutical composition comprising anyone of a CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragmentthereof or analogue thereof, apoptotic cells, or an apoptotic cellsupernatant, a tellurium-based compound, or an immune modulating agent,or any combination thereof, comprises multiple compositions, whereineach of the genetically modified immune cell, which in one embodiment isTCR T-cells, the CTLA-4 blocking agent, the alpha-1 anti-trypsin orfragment thereof or analogue thereof, the apoptotic cells, the apoptoticcell supernatant, the tellurium-based compound, or the immune modulatingagent, or any combination thereof, are comprised in a separatecomposition. In yet another embodiment, the composition comprising thegenetically modified immune cell, which in one embodiment is TCR T-cellsand the pharmaceutical composition comprising any one of a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, apoptotic cells, an apoptotic cell supernatant, atellurium-based compound, or an immune modulating agent, or anycombination thereof, comprises multiple compositions, wherein thegenetically modified immune cells, which in one embodiment are TCRT-cells, the CTLA-4 blocking agent, or the alpha-1 anti-trypsin orfragment thereof or analogue thereof, the tellurium-based compound, orthe immune modulating agent, or any combination thereof, or anycombination thereof are present in the genetically modified immune cell,for example a TCR T-cell, composition, and the apoptotic cells, or theapoptotic cell supernatant, are comprised in a separate composition.

In one embodiment, the composition comprising the genetically modifiedimmune cell, for example a dendritic cell and the pharmaceuticalcomposition comprising any one of a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment thereof or analogue thereof, apoptotic cells,or an apoptotic cell supernatant, a tellurium-based compound, or animmune modulating agent comprises a single composition. In anotherembodiment, the composition comprising the genetically modified immunecell, for example dendritic cells and the pharmaceutical compositioncomprising any one of a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, apoptotic cells, or anapoptotic cell supernatant, a tellurium-based compound, or an immunemodulating agent, or any combination thereof, comprises multiplecompositions, wherein each of the genetically modified immune cell,which in one embodiment is dendritic cells, the CTLA-4 blocking agent,the alpha-1 anti-trypsin or fragment thereof or analogue thereof, theapoptotic cells, the apoptotic cell supernatant, the tellurium-basedcompound, or the immune modulating agent, or any combination thereof,are comprised in a separate composition. In yet another embodiment, thecomposition comprising the genetically modified immune cell, which inone embodiment is dendritic cells and the pharmaceutical compositioncomprising any one of a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, apoptotic cells, an apoptoticcell supernatant, a tellurium-based compound, or an immune modulatingagent, or any combination thereof, comprises multiple compositions,wherein the genetically modified immune cells, which in one embodimentare dendritic cells, the CTLA-4 blocking agent, or the alpha-1anti-trypsin or fragment thereof or analogue thereof, thetellurium-based compound, or the immune modulating agent, or anycombination thereof, or any combination thereof are present in thegenetically modified immune cell, for example a dendritic cell,composition, and the apoptotic cells, or the apoptotic cell supernatant,are comprised in a separate composition.

In one embodiment, the composition comprising the genetically modifiedimmune cell, for example a NK cell and the pharmaceutical compositioncomprising any one of a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, apoptotic cells, or anapoptotic cell supernatant, a tellurium-based compound, or an immunemodulating agent comprises a single composition. In another embodiment,the composition comprising the genetically modified immune cell, forexample NK cells and the pharmaceutical composition comprising any oneof a CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment thereofor analogue thereof, apoptotic cells, or an apoptotic cell supernatant,a tellurium-based compound, or an immune modulating agent, or anycombination thereof, comprises multiple compositions, wherein each ofthe genetically modified immune cell, which in one embodiment is NKcells, the CTLA-4 blocking agent, the alpha-1 anti-trypsin or fragmentthereof or analogue thereof, the apoptotic cells, the apoptotic cellsupernatant, the tellurium-based compound, or the immune modulatingagent, or any combination thereof, are comprised in a separatecomposition. In yet another embodiment, the composition comprising thegenetically modified immune cell, which in one embodiment is NK cellsand the pharmaceutical composition comprising any one of a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, apoptotic cells, an apoptotic cell supernatant, atellurium-based compound, or an immune modulating agent, or anycombination thereof, comprises multiple compositions, wherein thegenetically modified immune cells, which in one embodiment are NK cells,the CTLA-4 blocking agent, or the alpha-1 anti-trypsin or fragmentthereof or analogue thereof, the tellurium-based compound, or the immunemodulating agent, or any combination thereof, or any combination thereofare present in the genetically modified immune cell, for example a NKcell, composition, and the apoptotic cells, or the apoptotic cellsupernatant, are comprised in a separate composition.

A skilled artisan would appreciate that a “pharmaceutical composition”may encompass a preparation of one or more of the active ingredientsdescribed herein with other chemical components such as physiologicallysuitable carriers and excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism.

A skilled artisan would appreciate that the phrases “physiologicallyacceptable carrier”, “pharmaceutically acceptable carrier”,“physiologically acceptable excipient”, and “pharmaceutically acceptableexcipient”, may be used interchangeably may encompass a carrier,excipient, or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered active ingredient.

A skilled artisan would appreciate that an “excipient” may encompass aninert substance added to a pharmaceutical composition to furtherfacilitate administration of an active ingredient. In one embodiment,excipients include calcium carbonate, calcium phosphate, various sugarsand types of starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

Techniques for formulation and administration of agents are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

In one embodiment, compositions are administered at the same time. In analternative embodiment, compositions are administered at differenttimes. In another embodiment, compositions comprising apoptotic cellsare administered prior to infusion or genetically modified immune cellsor receptors thereof. In another embodiment, compositions comprisingapoptotic cells are administered prior to CAR-T-cell infusion. Inanother embodiment, compositions comprising apoptotic cells areadministered prior to cytotoxic T-cell infusion. In another embodiment,compositions comprising apoptotic cells are administered prior tonatural killer cell infusion. In another embodiment, compositionscomprising apoptotic cells are administered prior to dendritic infusion.In another embodiment, compositions comprising apoptotic cells areadministered prior to infusion of a genetically modified T-cellreceptor.

In another embodiment, compositions comprising apoptotic cellsupernatants are administered prior to infusion or genetically modifiedimmune cells or receptors thereof. In another embodiment, compositionscomprising apoptotic cell supernatants are administered prior toCAR-T-cell infusion. In another embodiment, compositions comprisingapoptotic cell supernatants are administered prior to cytotoxic T-cellinfusion. In another embodiment, compositions comprising apoptotic cellsupernatants are administered prior to natural killer cell infusion. Inanother embodiment, compositions comprising apoptotic cell supernatantsare administered prior to dendritic infusion. In another embodiment,compositions comprising apoptotic cell supernatants are administeredprior to infusion of a genetically modified T-cell receptor.

In another embodiment, compositions comprising apoptotic cellsupernatants are administered prior to infusion of genetically modifiedimmune cells or receptors thereof. In another embodiment, compositionscomprising apoptotic cells are administered about 24 hours prior togenetically modified immune cell or receptor thereof infusion. Inanother embodiment, compositions comprising apoptotic cells areadministered about 24 hours prior to CAR T-cell, or cytotoxic T-cells,or natural killer cells, or dendritic cell or genetically modifiedT-cell receptor infusion. In another embodiment, compositions comprisingapoptotic cell supernatants are administered about 24 hours prior to CART-cell or cytotoxic T-cells, or natural killer cells, or dendritic cellor genetically modified T-cell receptor infusion. In another embodiment,compositions comprising apoptotic cells are administered about 2 hours,4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18hours 20 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72hours prior to CAR-T-cell or cytotoxic T-cells, or natural killer cells,or dendritic cell or genetically modified T-cell receptor infusion. Inanother embodiment, compositions comprising apoptotic cell supernatantsare administered about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12hours, 14 hours, 16 hours, 18 hours 20 hours, 22 hours, 24 hours, 36hours, 48 hours, 60 hours, or 72 hours prior to CAR T-cell or cytotoxicT-cells, or natural killer cells, or dendritic cell or geneticallymodified T-cell receptor infusion. Each possibility represents aseparate embodiment as disclosed herein.

In another embodiment, compositions comprising apoptotic cells areadministered after infusion of genetically modified immune cells orgenetically modified receptors thereof. In another embodiment,composition comprising apoptotic cells are administered after CAR-T-cellor cytotoxic T-cells, or natural killer cells, or dendritic cell orgenetically modified T-cell receptor infusion. In another embodiment,compositions comprising apoptotic cell supernatants are administeredafter infusion of genetically modified immune cells or geneticallymodified receptors thereof. In another embodiment, compositionscomprising apoptotic cell supernatants are administered after CAR T-cellor cytotoxic T-cells, or natural killer cells, or dendritic cell orgenetically modified T-cell receptor infusion. In another embodiment,compositions comprising apoptotic cells are administered about 24 hoursafter CAR-T-cell or cytotoxic T-cells, or natural killer cells, ordendritic cell or genetically modified T-cell receptor infusion. Inanother embodiment, compositions comprising apoptotic cells areadministered after infusion of genetically modified immune cells orgenetically modified receptors thereof. In another embodiment,compositions comprising apoptotic cell supernatants are administeredabout 24 hours after CAR T-cell or cytotoxic T-cells, or natural killercells, or dendritic cell or genetically modified T-cell receptorinfusion. In another embodiment, compositions comprising apoptotic cellsare administered about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12hours, 14 hours, 16 hours, 18 hours 20 hours, 22 hours, 24 hours, 36hours, 48 hours, 60 hours, or 72 hours after CAR-T-cell or cytotoxicT-cells, or natural killer cells, or dendritic cell or geneticallymodified T-cell receptor infusion. In another embodiment, compositionscomprising apoptotic cell supernatants are administered about 2 hours, 4hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18hours 20 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72hours after CAR T-cell or cytotoxic T-cells, or natural killer cells, ordendritic cell or genetically modified T-cell receptor infusion. Eachpossibility represents a separate embodiment as disclosed herein.

Formulations

Compositions disclosed herein comprising genetically modifiedimmunoresponsive cells or comprising the apoptotic cells or comprisingthe apoptotic cell supernatants, or any combination thereof, can beconveniently provided as sterile liquid preparations, e.g., isotonicaqueous solutions, suspensions, emulsions, dispersions, or viscouscompositions, which may be buffered to a selected pH, Liquidpreparations are normally easier to prepare than gels, other viscouscompositions, and solid compositions. Additionally, liquid compositionsare somewhat more convenient to administer, especially by injection.Viscous compositions, on the other hand, can be formulated within theappropriate viscosity range to provide longer contact periods withspecific tissues. Liquid or viscous compositions can comprise carriers,which can be a solvent or dispersing medium containing, for example,water, saline, phosphate buffered saline, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like) and suitablemixtures thereof.

Sterile injectable solutions can be prepared by incorporating thegenetically modified immunoresponsive cells or apoptotic cellsupernatants utilized in practicing the methods disclosed herein, in therequired amount of the appropriate solvent with various amounts of theother ingredients, as desired. Such compositions may be in admixturewith a suitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, dextrose, or the like. The compositionscan also be lyophilized. The compositions can contain auxiliarysubstances such as wetting, dispersing, or emulsifying agents (e.g.,methylcellulose), pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, colors, and the like,depending upon the route of administration and the preparation desired.Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17thedition, 1985, incorporated herein by reference, may be consulted toprepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. According to the disclosure herein,however, any vehicle, diluent, or additive used would have to becompatible with the genetically modified immunoresponsive cells or theirprogenitors.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thecompositions as disclosed herein may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride may be preferred particularly forbuffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose may be preferred because it is readily and economicallyavailable and is easy to work with.

Other suitable thickening agents include, for example, xanthan gum,carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and thelike. The preferred concentration of the thickener will depend upon theagent selected. The important point is to use an amount that willachieve the selected viscosity. Obviously, the choice of suitablecarriers and other additives will depend on the exact route ofadministration and the nature of the particular dosage form, e.g.,liquid dosage form (e.g., whether the composition is to be formulatedinto a solution, a suspension, gel or another liquid form, such as atime release form or liquid-filled form).

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert and will notaffect the viability or efficacy of the genetically modifiedimmunoresponsive cells as described in the methods disclosed herein.This will present no problem to those skilled in chemical andpharmaceutical principles, or problems can be readily avoided byreference to standard texts or by simple experiments (not involvingundue experimentation), from this disclosure and the documents citedherein.

One consideration concerning the therapeutic use of genetically modifiedimmunoresponsive cells disclosed herein is the quantity of cellsnecessary to achieve an optimal effect. The quantity of cells to beadministered will vary for the subject being treated. In a oneembodiment, between 10⁴ to 10¹⁰, between 10⁵ to 10⁹, or between 10⁶ and10⁸ genetically modified immunoresponsive cells disclosed herein areadministered to a human subject. More effective cells may beadministered in even smaller numbers. In some embodiments, at leastabout 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, and 5×10⁸ genetically modifiedimmunoresponsive cells disclosed herein are administered to a humansubject. The precise determination of what would be considered aneffective dose may be based on factors individual to each subject,including their size, age, sex, weight, and condition of the particularsubject. Dosages can be readily ascertained by those skilled in the artfrom this disclosure and the knowledge in the art.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods disclosed herein. Typically, any additives (inaddition to the active cell(s) and/or agent(s)) are present in an amountof 0.001 to 50% (weight) solution in phosphate buffered saline, and theactive ingredient is present in the order of micrograms to milligrams,such as about 0.0001 to about 5 wt %. In another embodiment about 0.0001to about 1 wt %. In still another embodiment, about 0.0001 to about 0.05wt % or about 0.001 to about 20 wt %. In a further embodiment, about0.01 to about 10 wt %. In another embodiment, about 0.05 to about 5 wt%. Of course, for any composition to be administered to an animal orhuman, and for any particular method of administration, it is preferredto determine therefore: toxicity, such as by determining the lethal dose(LD) and LD50 in a suitable animal model e.g., rodent such as mouse;and, the dosage of the composition(s), concentration of componentstherein and timing of administering the composition(s), which elicit asuitable response. Such determinations do not require undueexperimentation from the knowledge of the skilled artisan, thisdisclosure and the documents cited herein. And, the time for sequentialadministrations can be ascertained without undue experimentation.

Nucleic Acid Sequences, Vectors, Cells

In one embodiment, disclosed herein are an isolated nucleic acidsequence encoding a chimeric antigen receptor (CAR) as described hereinfor uses in the compositions and methods as disclosed herein.

In another embodiment, disclosed herein are a vector comprising thenucleic acid sequence encoding a chimeric antigen receptor (CAR) asdescribed herein.

In one embodiment, disclosed herein are an isolated nucleic acidsequence encoding a genetically modified T-cell receptor (TCR) asdescribed herein for uses in the compositions and methods as disclosedherein. In another embodiment, disclosed herein are a vector comprisingthe nucleic acid sequence encoding a genetically modified T-cellreceptor (TCR) as described herein.

Genetic modification of immunoresponsive cells (e.g., T-cells, CTLcells, NK cells) can be accomplished by transducing a substantiallyhomogeneous cell composition with a recombinant DNA construct. In oneembodiment, a retroviral vector (either gamma-retroviral or lentiviral)is employed for the introduction of the DNA construct into the cell. Forexample, a polynucleotide encoding a receptor that binds an antigen(e.g., a tumor antigen, or a valiant, or a fragment thereof), can becloned into a retroviral vector and expression can be driven from itsendogenous promoter, from the retroviral long terminal repeat, or from apromoter specific for a targeT-cell type of interest. Non-viral vectorsmay be used as well.

Non-viral approaches can also be employed for the expression of aprotein in cell. For example, a nucleic acid molecule can be introducedinto a cell by administering the nucleic acid in the presence oflipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413,1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al, Am.J. Med. Sci. 298:278, 1989; Staubinger et al, Methods in Enzymology101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al.,Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal ofBiological Chemistry 264:16985, 1989), or by micro-injection undersurgical conditions (Wolff et al., Science 247: 1465, 1990). Othernon-viral means for gene transfer include transfection in vitro usingcalcium phosphate, DEAE dextran, electroporation, and protoplast fusion.Liposomes can also be potentially beneficial for delivery of DNA into acell. Transplantation of normal genes into the affected tissues of asubject can also be accomplished by transferring a normal nucleic acidinto a cultivatable cell type ex vivo (e.g., an autologous orheterologous primary cell or progeny thereof), after which the cell (orits descendants) are injected into a targeted tissue or are injectedsystemically. Recombinant receptors can also be derived or obtainedusing transposases or targeted nucleases (e g Zinc finger nucleases,meganucleases, or TALE nucleases). Transient expression may be obtainedby RNA electroporation. cDNA expression for use in polynucleotidetherapy methods can be directed from any suitable promoter (e.g., thehuman cytomegalovirus (CMV), simian virus 40 (SV40), or metallothioneinpromoters), and regulated by any appropriate mammalian regulatoryelement or intron (e.g. the elongation factor 1aenhancer/promoter/intron structure). For example, if desired, enhancersknown to preferentially direct gene expression in specific cell typescan be used to direct the expression of a nucleic acid. The enhancersused can include, without limitation, those that are characterized astissue- or cell-specific enhancers. Alternatively, if a genomic clone isused as a therapeutic construct, regulation can be mediated by thecognate regulatory sequences or, if desired, by regulatory sequencesderived from a heterologous source, including any of the promoters orregulatory elements described above.

In another embodiment, disclosed herein are a cell comprising the vectorcomprising the nucleic acid sequence encoding a chimeric antigenreceptor (CAR) as disclosed herein.

Kits

In one embodiment, disclosed herein are a kit for inhibiting or reducingthe incidence of a cytokine release syndrome (CRS) or a cytokine stormin a subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) cancer therapy, the kit comprising a CAR T-cells and apoptoticcells as disclosed herein, either separately or pre-mixed.

In another embodiment, disclosed herein are a kit for inhibiting orreducing the incidence of a cytokine release syndrome (CRS) or acytokine storm in a subject undergoing chimeric antigenreceptor-expressing T-cell (CAR T-cell) cancer therapy, the kitcomprising a CAR T-cells and an apoptotic cell supernatant as disclosedherein, either separately or pre-mixed.

Disclosed herein are kits for inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm generated bytreatment or prevention of a neoplasia, pathogen infection, immunedisorder or allogeneic transplant, or by treating, preventing,inhibiting, reducing the incidence of, ameliorating, or alleviating acancer or a tumor. In one embodiment, the kit includes a therapeutic orprophylactic composition containing an effective amount of animmunoresponsive cells and apoptotic cells as disclosed herein in unitdosage form. In another embodiment, the kit includes a therapeutic orprophylactic composition containing an effective amount of animmunoresponsive cells and an apoptotic cell supernatant as disclosedherein in unit dosage form. In particular embodiments, the cells furthercomprise a co-stimulatory ligand. In another embodiment, kits furthercomprise an additional agent selected from the group comprising a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analogthereof, a tellurium-based compound, or an immune modulating agent, orany combination thereof. In some embodiments, the kit comprises asterile container which contains a therapeutic or prophylactic vaccine;such containers can be boxes, ampules, bottles, vials, tubes, bags,pouches, blister-packs, or other suitable container forms known in theart. Such containers can be made of plastic, glass, laminated paper,metal foil, or other materials suitable for holding medicaments.

If desired, the immunoresponsive cells and apoptotic cells or apoptoticcell supernatant are provided together with instructions foradministering the cells to a subject having or at risk of developing aneoplasia, pathogen infection, immune disorder or allogeneic transplantor tumors or cancer. The instructions will generally include informationabout the use of the composition for the treatment or prevention ofneoplasia, pathogen infection, immune disorder, allogeneic transplant,tumor or cancer. In other embodiments, the instructions include at leastone of the following: description of the therapeutic agent; dosageschedule and administration for treatment or prevention of a neoplasia,pathogen infection, immune disorder or allogeneic transplant, cancers,tumors, or symptoms thereof; precautions; warnings; indications;counter-indications; over dosage information; adverse reactions; animalpharmacology; clinical studies; and/or references. The instructions maybe printed directly on the container (when present), or as a labelapplied to the container, or as a separate sheet, pamphlet, card, orfolder supplied in or with the container.

A skilled artisan would appreciate that the term “antigen recognizingreceptor” may encompass a receptor that is capable of activating animmune cell (e.g., a T-cell) in response to antigen binding. Exemplaryantigen recognizing receptors may be native or endogenous T-cellreceptors or chimeric antigen receptors in which a tumor antigen-bindingdomain is fused to an intracellular signaling domain capable ofactivating an immune cell (e.g., a T-cell).

A skilled artisan would appreciate that the term “antibody” means notonly intact antibody molecules, but also fragments of antibody moleculesthat retain immunogen-binding ability. Such fragments are also wellknown in the art and are regularly employed both in vitro and in vivo.Accordingly, the skilled artisan would appreciate that the term“antibody” means not only intact immunoglobulin molecules but also thewell-known active fragments F(ab¹)2, and Fab. F(ab′)2₅ and Fab fragmentsthat lack the Fc fragment of intact antibody, clear more rapidly fromthe circulation, and may have less non-specific tissue binding of anintact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983). Theantibodies disclosed herein comprise whole native antibodies, bispecificantibodies; chimeric antibodies; Fab, Fab′, single chain V regionfragments (scFv), fusion polypeptides, and unconventional antibodies.

A skilled artisan would appreciate that the term “single-chain variablefragment” or “scFv” encompasses a fusion protein of the variable regionsof the heavy (VH) and light chains (VL) of an immunoglobulin covalentlylinked to form a VH::VL heterodimer. The heavy (VH) and light chains(VL) are either joined directly or joined by a peptide-encoding linker(e.g., 30, 15, 20, 25 amino acids), which connects the N-terminus of theVH with the C-terminus of the VL, or the C-terminus of the VH with theN-terminus of the VL, The linker is usually rich in glycine forflexibility, as well as serine or threonine for solubility. Despiteremoval of the constant regions and the introduction of a linker, scFvproteins retain the specificity of the original immunoglobulin. Singlechain Fv polypeptide antibodies can be expressed from a nucleic acidincluding VH- and VL-encoding sequences as described by Huston, et al.(Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Pat.Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent PublicationNos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitoryactivity have been described (see, e.g., Zhao et al., Hyrbidoma(Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcope is Muscle2012 Aug. 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli etal., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84;Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs havingstimulatory activity have been described (see, e.g., Peter et al., JBiol Chem 2003 25278(38):36740-7; Xie et al, Nat Biotech 1 9715(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 1 (5-6)-0.427-55;Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

By “affinity” is meant a measure of binding strength. Without beingbound to theory, affinity depends on the closeness of stereochemical fitbetween antibody combining sites and antigen determinants, on the sizeof the area of contact between them, and on the distribution of chargedand hydrophobic groups. Affinity also includes the term “avidity,” whichrefers to the strength of the antigen-antibody bond after formation ofreversible complexes. Methods for calculating the affinity of anantibody for an antigen are known in the art, including use of bindingexperiments to calculate affinity. Antibody activity in functionalassays (e.g., flow cytometry assay) is also reflective of antibodyaffinity. Antibodies and affinities can be phenotypically characterizedand compared using functional assays (e.g., flow cytometry assay).

A skilled artisan would appreciate that the term “chimeric antigenreceptor” or “CAR” may encompass an antigen-binding domain that is fusedto an intracellular signaling domain capable of activating orstimulating an immune cell. In one embodiment, the CAR's extracellularbinding domain is composed of a single chain variable fragment (scFv)derived from fusing the variable heavy and light regions of a murine orhumanized monoclonal antibody. Alternatively, scFvs may be used that arederived from Fab's (instead of from an antibody, e.g., obtained from Fablibraries), in various embodiments, this scFv is fused to atransmembrane domain and then to an intracellular signaling domain. Invarious embodiments, the CAR is selected to have high affinity oravidity for the antigen.

Polypeptides and Analogs

Also included in the methods disclosed herein are anti-MUC1, CD28, CD3ζ,and various scFv polypeptides or fragments thereof that are modified inways that enhance their anti-neoplastic activity (e.g., a humanizedmonoclonal antibody) when expressed in an immunoresponsive cell. Incertain embodiments, the methods disclosed herein comprise optimizing anamino acid sequence or nucleic acid sequence by producing an alterationin the sequence. Such alterations may include certain mutations,deletions, insertions, or post-translational modifications. Thedisclosure provided herein further includes analogs of anynaturally-occurring polypeptide disclosed herein. Analogs can differfrom a naturally-occurring polypeptide disclosed herein by amino acidsequence differences, by post-translational modifications, or by both.Analogs disclosed herein will generally exhibit at least 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%>, 99% or more identity with all orpart of a naturally-occurring amino, acid sequence disclosed herein. Thelength of sequence comparison is at least 5, 10, 15 or 20 amino acidresidues. In another embodiment, at least 25, 50, or 75 amino acidresidues. In still another embodiment, more than 100 amino acidresidues. Again, in an exemplary approach to determining the degree ofidentity, a BLAST program may be used, with a probability score betweene″3 and e″100 indicating a closely related sequence. Modificationsinclude in vivo and in vitro chemical derivatization of polypeptides,e.g., acetylation, carboxylation, phosphorylation, or glycosylation;such modifications may occur during polypeptide synthesis or processingor following treatment with isolated modifying enzymes. Analogs can alsodiffer from the naturally-occurring polypeptides disclosed herein byalterations in primary sequence. These include genetic variants, bothnatural and induced (for example, resulting from random mutagenesis byirradiation or exposure to ethanemethyl sulfate or by site-specificmutagenesis as described in Sambrook, Fritsch and Maniatis, MolecularCloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel etal, supra). Also included are cyclized peptides, molecules, and analogswhich contain residues other than L-amino acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., beta (β) orgamma (γ) amino acids.

Non-protein analogs have a chemical structure designed to mimic thefunctional activity of a protein disclosed herein. Such analogs areadministered according to methods disclosed herein. Such analogs mayexceed the physiological activity of the original polypeptide. Methodsof analog design are well known in the art, and synthesis of analogs canbe carried out according to such methods by modifying the chemicalstructures such that the resultant analogs increase the antineoplasticactivity of the original polypeptide when expressed in animmunoresponsive cell. These chemical modifications include, but are notlimited to, substituting alternative R groups and varying the degree ofsaturation at specific carbon atoms of a reference polypeptide. Inanother embodiment, the protein analogs are relatively resistant to invivo degradation, resulting in a more prolonged therapeutic effect uponadministration. Assays for measuring functional activity include, butare not limited to, those described in the Examples below.

The term “immunosuppressive activity” describes induction of signaltransduction or changes in protein expression in a cell (e.g., anactivated immunoresponsive cell) resulting in a decrease in an immuneresponse. Polypeptides known to suppress or decrease an immune responsevia their binding include CD47, PD-1, CTLA-4, and their correspondingligands, including SIRPa, PD-L1, PD-L2, B7-1, and B7-2. Suchpolypeptides are present in the tumor microenvironment and inhibitimmune responses to neoplastic cells. In various embodiments,inhibiting, blocking, or antagonizing the interaction ofimmunosuppressive polypeptides and/or their ligands enhances the immuneresponse of the immunoresponsive cell.

The term “immunostimulatory activity” describes induction of signaltransduction or changes in protein expression in a cell (e.g., anactivated immunoresponsive cell) resulting in an increased immuneresponse Immunostimulatory activity may include pro-inflammatoryactivity. Polypeptides known to stimulate or increase an immune responsevia their binding include CD28, OX-40, 4-IBB, and their correspondingligands, including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides arepresent in the tumor microenvironment and activate immune responses toneoplastic cells. In various embodiments, promoting, stimulating, oragonizing pro-inflammatory polypeptides and/or their ligands enhancesthe immune response of the immunoresponsive cell.

Nucleic acid molecules useful in the methods disclosed herein includeany nucleic acid molecule that encodes a polypeptide disclosed herein ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; immel, A.R. (1987) Methods Enzymol. 152:507).

A skilled artisan would appreciate that the term “substantiallyidentical” may encompass a polypeptide or nucleic acid moleculeexhibiting at least 50% identity to a reference amino acid sequence (forexample, any one of the amino acid sequences described herein) ornucleic acid sequence (for example, any one of the nucleic acidsequences described herein). In one embodiment, such a sequence is atleast 60%, 80% or 85%, 90%, 95% or even 99% identical at the amino acidlevel or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e-3 and e-100 indicating a closely related sequence.

A skilled artisan would appreciate that the term “analog” may encompassa structurally related polypeptide or nucleic acid molecule having thefunction of a reference polypeptide or nucleic acid molecule.

A skilled artisan would appreciate that the term “ligand” may encompassa molecule that binds to a receptor. In particular, the ligand binds areceptor on another cell, allowing for cell-to-cell recognition and/orinteraction.

A skilled artisan would appreciate that the term “constitutiveexpression” may encompass expression under all physiological conditions.

A skilled artisan would appreciate that the term “disease” ay encompassany condition or disorder that damages or interferes with the normalfunction of a cell, tissue, or organ. Examples of diseases includeneoplasia or pathogen infection of cell.

A skilled artisan would appreciate that the term “effective amount” mayencompass an amount sufficient to have a therapeutic effect. In oneembodiment, an “effective amount” is an amount sufficient to arrest,ameliorate, or inhibit the continued proliferation, growth, ormetastasis (e.g., invasion, or migration) of a neoplasia.

A skilled artisan would appreciate that the term “neoplasia” mayencompass a disease characterized by the pathological proliferation of acell or tissue and its subsequent migration to or invasion of othertissues or organs. Neoplasia growth is typically uncontrolled andprogressive, and occurs under conditions that would not elicit, or wouldcause cessation of, multiplication of normal cells. Neoplasias canaffect a variety of cell types, tissues, or organs, including but notlimited to an organ selected from the group consisting of bladder, bone,brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder,heart, intestines, kidney, liver, lung, lymph node, nervous tissue,ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen,stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter,urethra, uterus, and vagina, or a tissue or cell type thereof.Neoplasias include cancers, such as sarcomas, carcinomas, orplasmacytomas (malignant tumor of the plasma cells).

A skilled artisan would appreciate that the term “pathogen” mayencompass a virus, bacteria, fungi, parasite or protozoa capable ofcausing disease.

A skilled artisan would appreciate that the term “tumor antigen” or“tumor associated antigen” may encompass an antigen (e.g., apolypeptide) that is uniquely or differentially expressed on a tumorcell compared to a normal or non-IS neoplastic cell. With reference tothe compositions and methods disclosed herein, a tumor antigen includesany polypeptide expressed by a tumor that is capable of activating orinducing an immune response via an antigen recognizing receptor (e.g.,CD 19, MUCI) or capable of suppressing an immune response viareceptor-ligand binding (e.g., CD47, PD-L1/L2, B7.1/2).

A skilled artisan would appreciate that the term “virus antigen” mayencompass a polypeptide expressed by a virus that is capable of inducingan immune response.

The terms “comprises”, “comprising”, and are intended to have the broadmeaning ascribed to them in U.S. Patent Law and can mean “includes”,“including” and the like. Similarly, the term “consists of” and“consists essentially of” have the meanings ascribed to them in U.S.Patent Law. The compositions and methods as disclosed herein areenvisioned to either comprise the active ingredient or specified step,consist of the active ingredient or specified step, or consistessentially of the active ingredient or specified step.

A skilled artisan would appreciate that the term “treatment” mayencompass clinical intervention in an attempt to alter the diseasecourse of the individual or cell being treated, and can be performedeither for prophylaxis or during the course of clinical pathology.Therapeutic effects of treatment include, without limitation, preventingoccurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastases, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. By preventing progression of a diseaseor disorder, a treatment can prevent deterioration due to a disorder inan affected or diagnosed subject or a subject suspected of having thedisorder, but also a treatment may prevent the onset of the disorder ora symptom of the disorder in a subject at risk for the disorder orsuspected of having the disorder.

A skilled artisan would appreciate that the term “subject” may encompassa vertebrate, in one embodiment, to a mammal, and in one embodiment, toa human Subject may also refer, in one embodiment, to domesticated suchas cows, sheep, horses, cats, dogs and laboratory animals such as mice,rats, gerbils, hamsters, etc.

In one embodiment, disclosed herein are CAR T-cells in which the CAR isdirected to a peptide of interest. In one embodiment, the CAR binds to apeptide of interest. In another embodiment, the CAR targets a peptide ofinterest. In another embodiment, the CAR activates a peptide ofinterest. In another embodiment, the CAR is a ligand of the peptide ofinterest. In another embodiment, the peptide of interest is a ligand ofthe CAR. Each of these embodiments is to be considered part disclosedherein.

In one embodiment, the immune cell as disclosed herein is not a T-cell.In another embodiment, the immune cell as disclosed herein is not an NKcell. In another embodiment, the immune cell as disclosed herein is nota CTL. In another embodiment, the immune cell as disclosed herein is nota regulatory T-cell. In another embodiment, the immune cell is not ahuman embryonic stem cell. In another embodiment, the immune cell is nota pluripotent stem cell from which lymphoid cells may be differentiated.

Methods of Use

One approach to immunotherapy involves engineering a patient's ownimmune cells to create genetically modified immune cells that willrecognize and attack their tumor Immune cells are collected andgenetically modified, as described herein, for example to producechimeric antigen receptors (CAR) on their cell surface that will allowthe immune cell, for example a T-cell, to recognize a specific proteinantigen on a tumor or cancer cell. An expanded population of geneticallymodified immune cells, for example CAR T-cells, is then administered tothe patient. In one embodiment, the administered cells multiply in thepatient's body and recognize and kill cancer and tumor cells that harborthe antigen on their surface. In another embodiment, the administeredcells multiply in a patient's body and recognize and killtumor-associated antigens, which leads to the death of cancer and tumorcells.

In one embodiment, disclosed herein are methods of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing CAR T-cell cancer therapy, and methods ofdecreasing or inhibiting cytokine production in a subject experiencingcytokine release syndrome or cytokine storm, said methods comprising thestep of administering a composition comprising apoptotic cells or asupernatant of apoptotic cells. In another embodiment, disclosed hereinare methods of treating cytokine release syndrome or cytokine storm in asubject undergoing CAR T-cell cancer therapy. In another embodiment,disclosed herein are methods of preventing cytokine release syndrome orcytokine storm in a subject undergoing CAR T-cell cancer therapy. Inanother embodiment, disclosed herein are methods of alleviating cytokinerelease syndrome or cytokine storm in a subject undergoing CAR T-cellcancer therapy. In another embodiment, disclosed herein are methods ofameliorating cytokine release syndrome or cytokine storm in a subjectundergoing CAR T-cell cancer therapy. In another embodiment,administration of apoptotic cells or an apoptotic supernatant orcompositions thereof does not reduce the efficacy of the CAR T-celltherapy.

In one embodiment, disclosed herein are methods of inhibiting orreducing the incidence of a cytokine release syndrome (CRS) or acytokine storm in a subject undergoing chimeric antigenreceptor-expressing T-cell (CAR T-cell) cancer therapy, wherein themethod comprises the step of administering a composition comprisingapoptotic cells or an apoptotic cell supernatant or compositions thereofto said subject. In another embodiment, inhibiting or reducing theincidence of a cytokine release syndrome (CRS) or a cytokine storm isdetermined by measuring cytokine levels in a subject undergoing chimericantigen receptor-expressing T-cell cancer therapy and being administeredapoptotic cells or an apoptotic supernatant. In another embodiment,measured levels of cytokines are compared with cytokine levels in asubject not undergoing CAR T-cell cancer therapy. In another embodiment,measured cytokine levels are compared with cytokine levels in a subjectnot administer apoptotic cells or an apoptotic supernatant. In yetanother embodiment, measured cytokine levels are compared with a controlsubject.

In another embodiment, the level of pro-inflammatory cytokines arereduced in the subject compared with a subject undergoing CAR T-cellcancer therapy and not administered said apoptotic cells or saidapoptotic cell supernatant or compositions thereof. In anotherembodiment, methods disclosed herein reduce or inhibit the level ofproduction of at least one pro-inflammatory cytokines compared with asubject undergoing CAR T-cell cancer therapy and not administered saidapoptotic cells or said apoptotic cell supernatant or compositionsthereof.

In another embodiment, a method disclosed herein may further compriseadministration of additional agents. Alternatively, a method disclosedherein may comprise administration of additional agents and notapoptotic cells or an apoptotic cell supernatant. In still a furtherembodiment, additional agents may be those compounds or compositionsthat enhance or improve, or any combination thereof, CAR T-cell cancertherapy. In yet a further embodiment, additional agents that enhance orimprove CAR T-cell cancer therapy include CTLA-4 blocking agents, analpha-1 anti-trypsin or functional fragment thereof, or an analoguethereof, a tellurium-based compound, or an immune-modulating agent, orany combination thereof. In another embodiment, an additional agentincludes apoptotic cells or an apoptotic supernatant. In anotherembodiment, administration of an additional agent, a described herein,is prior to, concurrent with, of following said CAR T-cell cancertherapy.

In one embodiment, an IL-6 receptor antagonist, which in one embodimentis tocilizumab is used with the compositions and methods as disclosedherein.

In one embodiment, adoptively transferred T-cells engraft and expandmore efficiently in a lymphopenic host. Thus, in one embodiment, thesubject is subjected to lymphodepletion prior to transfer of CAR T-cellsor other modified immune cells. In another embodiment, the subjectreceiving the CAR T-cells is given T-cell-supportive cytokines.

In one embodiment, the T-cells are effector T-cells. In anotherembodiment, the T-cells are naïve T-cells. In another embodiment, theT-cells are central memory (T_(CM)) T-cells. In another embodiment, theT-cells are Th17 cells. In another embodiment, the T-cells are T stemmemory cells. In another embodiment, the T-cells have high replicativecapacity. In another embodiment, T-cell expansion occurs in the patient.In another embodiment, small numbers of cells may be transferred to apatient. In another embodiment, T-cell expansion occurs in vitro. Inanother embodiment, large numbers of cells may be transferred to apatient, cells and/or supernatants may be transferred to a patient onmultiple occasions, or a combination thereof.

In one embodiment, an advantage of CAR T-cells is that because they arespecific for cell-surface molecules, they overcome the constraints ofMHC-restricted TCR recognition and avoid tumor escape throughimpairments in antigen presentation or human leukocyte antigenexpression.

In one embodiment, disclosed herein is a method of reducing a tumorburden in a subject, said method comprising the step of administering tosaid subject any of the compositions as described herein.

In one embodiment, reducing the tumor burden comprises reducing thenumber of tumor cells in the subject. In another embodiment, reducingthe tumor burden comprises reducing tumor size in the subject. Inanother embodiment, reducing the tumor burden comprises eradicating thetumor in the subject.

In another embodiment, disclosed herein is a method of inducing tumorcell death in a subject, said method comprising the step ofadministering to said subject any of the compositions as describedherein. In another embodiment, a method as disclosed herein for inducingtumor cell death in a subject comprises administering immune cells, suchas NK cells or T-cells comprising engineered chimeric antigen receptorswith at least an additional agent to decrease toxic cytokine release or“cytokine release syndrome” (CRS) or “severe cytokine release syndrome”(sCRS) or “cytokine storm” in the subject.

In another embodiment, disclosed herein is a method of increasing orlengthening the survival of a subject having neoplasia, comprising thestep of administering to said subject any of the compositions asdescribed herein. In another embodiment, a method of increasing orlengthening the survival of a subject comprises administering immunecells, such as NK cells or T-cells comprising engineered chimericantigen receptors with at least an additional agent to decrease toxiccytokine release or “cytokine release syndrome” (CRS) or “severecytokine release syndrome” (sCRS) or “cytokine storm” in the subject.

In another embodiment, disclosed herein is a method of increasing orlengthening the survival of a subject having neoplasia, comprising thestep of administering to said subject any of the compositions asdescribed herein.

In another embodiment, disclosed herein is a method of preventingneoplasia in a subject, said method comprising the step of administeringto said subject any of the compositions as described herein.

In one embodiment, the neoplasia is selected from the group consistingof blood cancer, B cell leukemia, multiple myeloma, lymphoblasticleukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma,ovarian cancer, or a combination thereof.

In another embodiment, disclosed herein is a method of treating bloodcancer in a subject in need thereof, comprising the step ofadministering to said subject any of the compositions as describedherein. In one embodiment, the blood cancer is selected from the groupconsisting of B cell leukemia, multiple myeloma, acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin'slymphoma.

In one embodiment, a method of decreasing or inhibiting cytokineproduction in a subject experiencing cytokine release syndrome orcytokine storm or vulnerable to a cytokine release syndrome or cytokinestorm, as disclosed herein, decreases or inhibits cytokine production.In another embodiment, the method decreases or inhibits pro-inflammatorycytokine production. In a further embodiment, the method decreases orinhibits at least one pro-inflammatory cytokine. In another embodiment,wherein the subject is undergoing CAR T-cell cancer therapy, the methoddoes not reduce the efficacy of the CAR T-cell therapy.

The methods provided herein comprise administering a T-cell, NK cell, orCTL cell disclosed herein, in an amount effective to achieve the desiredeffect, be it palliation of an existing condition or prevention ofrecurrence. For treatment, the amount administered is an amounteffective in producing the desired effect. An effective amount can beprovided in one or a series of administrations. An effective amount canbe provided in a bolus or by continuous perfusion.

A skilled artisan would recognize that an “effective amount” (or,“therapeutically effective amount”) may encompass an amount sufficientto effect a beneficial or desired clinical result upon treatment. Aneffective amount can be administered to a subject in one or more doses.In terms of treatment, an effective amount is an amount that issufficient to palliate, ameliorate, stabilize, reverse or slow theprogression of the disease, or otherwise reduce the pathologicalconsequences of the disease. The effective amount is generallydetermined by the physician on a case-by-case basis and is within theskill of one in the art. Several factors are typically taken intoaccount when determining an appropriate dosage to achieve an effectiveamount. These factors include age, sex and weight of the subject, thecondition being treated, the severity of the condition and the form andeffective concentration of the antigen-binding fragment administered.

For adoptive immunotherapy using antigen-specific T-cells, for exampleCAR T-cells, cell doses in the range of 10⁶-10¹⁰ (e.g., 10⁹) aretypically infused. Upon administration of the genetically modified cellsinto the host and subsequent differentiation, T-cells are induced thatare specifically directed against the specific antigen. “Induction” ofT-cells may include inactivation of antigen-specific T-cells such as bydeletion or anergy. Inactivation is particularly useful to establish orreestablish tolerance such as in autoimmune disorders. The modifiedcells can be administered by any method known in the art including, butnot limited to, intravenous, subcutaneous, intranodal, intratumoral,intrathecal, intrapleural, intraperitoneal and directly to the thymus.In one embodiment, the T-cells are not administered intraperitoneally.In one embodiment, the T-cells are administered intratumorally.

Compositions comprising genetically modified immunoresponsive cells asdisclosed herein (e.g., T-cells, N cells, CTL cells, or theirprogenitors) can be provided systemically or directly to a subject forthe treatment of a neoplasia, pathogen infection, or infectious disease.In one embodiment, cells disclosed herein are directly injected into anorgan of interest (e.g., an organ affected by a neoplasia).Alternatively, compositions comprising genetically modifiedimmunoresponsive cells are provided indirectly to the organ of interest,for example, by administration into the circulatory system (e.g., thetumor vasculature). Expansion and differentiation agents can be providedprior to, during or after administration of the cells to increaseproduction of T-cells, NK cells, or CTL cells in vitro or in vivo.

The modified cells can be administered in any physiologically acceptablevehicle, normally intravascularly, although they may also be introducedinto bone or other convenient site where the cells may find anappropriate site for regeneration and differentiation (e.g., thymus).Usually, at least 1×10⁵ cells will be administered, eventually reaching1×10¹⁰ or more. Genetically modified immunoresponsive cells disclosedherein may comprise a purified population of cells. Those skilled in theart can readily determine the percentage of genetically modifiedimmunoresponsive cells in a population using various well-known methods,such as fluorescence activated cell sorting (FACS). In some embodiments,ranges of purity in populations comprising genetically modifiedimmunoresponsive cells are about 50 to about 55%, about 55 to about 60%,and about 65 to about 70%. In other embodiments, the purity is about 70to about 75%, about 75 to about 80%, about 80 to about 85%. In furtherembodiments, the purity is about 85 to about 90%, about 90 to about 95%,and about 95 to about 100%. Dosages can be readily adjusted by thoseskilled in the art (e.g., a decrease in purity may require an increasein dosage). The cells can be introduced by injection, catheter, or thelike. If desired, factors can also be included, including, but notlimited to, interleukins, e.g. IL-2, IL-3, IL-6, IL-11, IL7, IL12, ILIS,IL21, as well as the other interleukins, the colony stimulating factors,such as G-, M- and GM-CSF, interferons, e.g. gamma-interferon anderythropoietin.

Compositions include pharmaceutical compositions comprising geneticallymodified immunoresponsive cells or their progenitors and apharmaceutically acceptable carrier. Administration can be autologous orheterologous. For example, immunoresponsive cells, or progenitors can beobtained from one subject, and administered to the same subject or adifferent, compatible subject. Peripheral blood derived immunoresponsivecells disclosed herein or their progeny (e.g., in vivo, ex vivo or invitro derived) can be administered via localized injection, includingcatheter administration, systemic injection, localized injection,intravenous injection, or parenteral administration. When administeringa therapeutic composition as disclosed herein (e.g., a pharmaceuticalcomposition containing a genetically modified immunoresponsive cell), itwill generally be formulated in a unit dosage injectable form (solution,suspension, emulsion).

In another embodiment, disclosed herein is a method of producing acomposition comprising CAR T-cells or other immune cells as disclosedherein and apoptotic cells or an apoptotic cell supernatant, the methodcomprising introducing into the T-cell or immune cell the nucleic acidsequence encoding the CAR that binds to an antigen of interest. In analternative embodiment, the compositions comprising CAR T-cells or otherimmune cells as disclosed herein are separate from the compositioncomprising apoptotic cells or an apoptotic supernatant.

A skilled artisan would appreciate that an anti-tumor immunity responseelicited by the genetically modified immune cells, for exampleCAR-modified T cells, may be an active or a passive immune response. Inaddition, the CAR mediated immune response may be part of an adoptiveimmunotherapy approach in which CAR-modified T-cells induce an immuneresponse specific to the antigen binding moiety in the CAR.

A skilled artisan would appreciate that immunotherapeutics may encompassthe use of immune effector cells and molecules to target and destroycancer cells. The immune effector may be, for example, an antibodyspecific for some marker on the surface of a tumor cell. The antibodyalone may serve as an effector of therapy or it may recruit other cellsto actually effect cell killing. The antibody also may be conjugated toa drug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and serve merely as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target. Various effector cells include cytotoxic T cells and NKcells.

Malignancies

In some embodiments, CAR T-cells are utilized in methods of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor wherein the methods comprise the step ofadministering chimeric antigen receptor-expressing T-cells (CART-cells). As disclosed herein, these methods may further compriseadministering an additional agent in an effort to inhibit or decreasethe incidence of CRS or cytokine storm.

In one embodiment, the cancer is a B-cell malignancy. In one embodiment,the B-cell malignancy is leukemia. In another embodiment, the B-cellmalignancy is acute lymphoblastic leukemia (ALL) In another embodiment,the B-cell malignancy is chronic lymphocytic leukemia.

In one embodiment, the cancer is leukemia. In one embodiment, the canceris lymphoma. In one embodiment, the lymphoma is large B-cell lymphoma.

In one embodiment, the tumor is a solid tumor. In another embodiment, asolid tumor is an abnormal mass of tissue lacking cysts or liquid areas.In another embodiment, solid tumors are neoplasms (new growth of cells)or lesions (damage of anatomic structures or disturbance ofphysiological functions) formed by an abnormal growth of body tissuecells other than blood, bone marrow or lymphatic cells. In anotherembodiment, a solid tumor consists of an abnormal mass of cells whichmay stem from different tissue types such as liver, colon, breast, orlung, and which initially grows in the organ of its cellular origin.However, such cancers may spread to other organs through metastatictumor growth in advanced stages of the disease.

In one embodiment, the tumor is a solid tumor. In another embodiment,examples of solid tumors are sarcomas, carcinomas, and lymphomas.

In another embodiment, the solid tumor comprises an Adrenocortical Tumor(Adenoma and Carcinoma), a Carcinoma, a Colorectal Carcinoma, a DesmoidTumor, a Desmoplastic Small Round Cell Tumor, an Endocrine Tumor, anEwing Sarcoma, a Germ Cell Tumor, a Hepatoblastoma a HepatocellularCarcinoma, a Melanoma, a Neuroblastoma, an Osteosarcoma, aRetinoblastoma, a Rhabdomyosarcoma, a Soft Tissue Sarcoma Other ThanRhabdomyosarcoma, and a Wilms Tumor. In one embodiment, the solid tumoris a breast tumor. In another embodiment, the solid tumor is a prostatecancer. In another embodiment, the solid tumor is a colon cancer. In oneembodiment, the tumor is a brain tumor. In another embodiment, the tumoris a pancreatic tumor. In another embodiment, the tumor is a colorectaltumor.

In another embodiment, compositions and methods as disclosed herein havetherapeutic and/or prophylactic efficacy against sarcomas and carcinomas(e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, uterine cancer, testicular cancer, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma,schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).The compositions and methods as disclosed herein may be used to treat,prevent, inhibit, ameliorate, reduce the incidence of, or alleviate anysolid tumor known in the art.

In another embodiment, the tumor is a hematological tumor. In oneembodiment, hematological tumors are cancer types affecting blood, bonemarrow, and lymph nodes. Hematological tumors may derive from either ofthe two major blood cell lineages: myeloid and lymphoid cell lines. Themyeloid cell line normally produces granulocytes, erythrocytes,thrombocytes, macrophages, and masT-cells, whereas the lymphoid cellline produces B, T, NK and plasma cells. Lymphomas (e.g. Hodgkin'sLymphoma), lymphocytic leukemias, and myeloma are derived from thelymphoid line, while acute and chronic myelogenous leukemia (AML, CML),myelodysplastic syndromes and myeloproliferative diseases are myeloid inorigin.

In another embodiment, compositions and methods as disclosed herein havetherapeutic and/or prophylactic efficacy against leukemias (e.g., acuteleukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acutemyeloblastic leukemia, acute promyelocyte leukemia, acute myelomonocyticleukemia, acute monocytic leukemia, acute erythroleukemia, chronicleukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia),polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease),Waldenstrom's macroglobulinemia, heavy chain disease. The compositionsand methods as disclosed herein may be used to treat, prevent, inhibit,ameliorate, reduce the incidence of, or alleviate any hematologicaltumor known in the art.

In one embodiment, disclosed herein are active fragments of any one ofthe polypeptides or peptide domains disclosed herein. A skilled artisanwould appreciate that the term “a fragment” may encompass at least 5,10, 13, or 15 amino acids. In other embodiments a fragment is at least20 contiguous amino acids. Fragments disclosed herein can be generatedby methods known to those skilled in the art or may result from normalprotein processing (e.g., removal of amino acids from the nascentpolypeptide that are not required for biological activity or removal ofamino acids by alternative mRNA splicing or alternative proteinprocessing events).

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and specifically refer to a polyclonal antibody, amonoclonal antibody, or any fragment thereof, which retains the bindingactivity of the antibody. In certain embodiments, methods disclosedherein comprise use of a chimeric antibody, a humanized antibody, or ahuman antibody.

A skilled artisan would appreciate that the term “polyclonal antibody(or antibodies)” may encompass a population of different antibodiesdirected against different determinants (epitopes) of the same antigen.

A skilled artisan would appreciate that the term “monoclonal antibody(or antibodies)” may encompass a population of substantially homogenousantibodies, i.e., the individual antibodies comprising the populationare identical except for possibly naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are directed againsta single antigenic site.

The monoclonal antibodies disclosed herein can be made using thehybridoma method first described by Kohler et al, Nature, 256: 495(1975), or may be made by recombinant DNA methods (e.g. U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies to the protein of interestgenerally are raised in animals by subcutaneous (sc) or intraperitoneal(ip) injections of the desired protein of interest and an adjuvant. Inone embodiment, the animals are immunized with the protein of interestcoupled to Keyhole limpet hemocyanin (KLH, Sigma Aldrich) as a carrierprotein.

The protein of interest used for animal immunization are prepared usingmethods well-known in the art. For example, the protein of interest maybe produced by recombinant methods or by peptide synthesis methods.

Alternatively, lymphocytes may be immunized in vitro and then fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal Biochem.,107: 220 (1980).

The antibodies disclosed herein can be produced by using combinatoriallibraries to screen for synthetic antibody clones with the desiredactivity. In principle, synthetic antibody clones are selected byscreening phage libraries containing phage that display variousfragments of antibody variable region (Fv) fused to phage coat proteinusing methods well known in the art.

A skilled artisan would appreciate that the term “any fragment thereofwhich retains the binding activity of the antibody” may encompass aportion of an antibody, which may comprise the antigen-binding orvariable region thereof, which is capable of binding to the targetantigen of the intact antibody. Examples of antibody fragments includeFab, Fab′, F(ab′)₂, and Fv fragments.

These antibody fragments may be generated by recombinant techniques orby traditional means, such as enzymatic digestion. Papain digestion of 6antibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single binding site, and a residual “Fc”fragment. Pepsin treatment yields an F(ab′)₂, fragment that has twoantigen-combining sites and is still capable of cross-linking antigen.“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and binding site.

The polyclonal antibodies and the monoclonal antibodies disclosed hereinare prepared using methods well known in the art.

In one embodiment, disclosed herein are a CAR T-cell or relatedcomposition in which the CAR is endogenous to the T-cell. In oneembodiment, “endogenous” comprises a nucleic acid molecule (e.g., acDNA, DNA or RNA molecule) or polypeptide that is normally expressed ina cell or tissue.

In another embodiment, disclosed herein are a CAR T-cell or relatedcomposition in which the CAR is exogenous to the T-cell. In oneembodiment, “exogenous” comprises a nucleic acid molecule or polypeptidethat is not endogenously present in the cell, or not present at a levelsufficient to achieve the functional effects obtained when artificiallyover-expressed. A skilled artisan would appreciate that the term“exogenous” would therefore encompass any recombinant nucleic acidmolecule or polypeptide expressed in a cell, such as foreign,heterologous, and over-expressed nucleic acid molecules andpolypeptides.

In one embodiment, disclosed herein are immune cells, in one embodiment,CAR T-cells in which the T-cell is autologous to the subject. In anotherembodiment, the CAR T-cells are heterologous to the subject. In oneembodiment, the CAR T-cells are allogeneic. In one embodiment, the CART-cells are universal allogeneic CAR T-cells. In another embodiment, theT-cells may be autologous, allogeneic, or derived in vitro fromengineered progenitor or stem cells.

In another embodiment, the CAR T-cells and apoptotic cells describedherein, are both derived from the same source. In a further embodiment,the CAR T-cells and apoptotic cells described herein, are both derivedfrom the subject (FIG. 1). In an alternative embodiment, the CAR T-cellsand apoptotic cells described herein, are derived from differentsources. In yet another embodiment, the CAR T-cells are autologous andthe apoptotic cells described herein, are allogeneic (FIG. 2). A skilledartisan would appreciate that similarly, an apoptotic cell supernatantmay be made from cells derived from the same source as the CAR T-cell,which may in one embodiment be autologous cells, or an apoptotic cellsupernatant may be made from cells derived from a source different fromthe source of CAR T-cells.

A skilled artisan would appreciate that the term “heterologous” mayencompass a tissue, cell, nucleic acid molecule or polypeptide that isderived from a different organism. In one embodiment, a heterologousprotein is a protein that was initially cloned from or derived from adifferent T-cell type or a different species from the recipient and thatis not normally present in a cell or sample obtained from a cell.

A skilled artisan would appreciate that the term “autologous” mayencompass a tissue, cell, nucleic acid molecule or polypeptide in whichthe donor and recipient is the same person.

A skilled artisan would appreciate that the term “allogeneic” mayencompass a tissue, cell, nucleic acid molecule or polypeptide that isderived from separate individuals of the same species. In oneembodiment, allogeneic donor cells are genetically distinct from therecipient.

In another embodiment, compositions and methods as disclosed hereinutilize combination therapy with apoptotic cells or apoptoticsupernatants as disclosed herein, and one or more CTLA-4-blocking agentssuch as Ipilimumab. In one embodiment, CTLA-4 is a potent inhibitor ofT-cell activation that helps to maintain self-tolerance. In oneembodiment, administration of an anti-CTLA-4 blocking agent, which inanother embodiment, is an antibody, produces a net effect of T-cellactivation. In another embodiment, compositions and methods as disclosedherein utilize combined therapy comprising apoptotic cells, CAR T-cells,and one or more CTLA-4-blocking agents.

In some cases, a polypeptide of and for use in the methods as disclosedherein comprises at least one conservative amino acid substitutionrelative to an unmodified amino acid sequence. In other cases, thepolypeptide comprises a non-conservative amino acid substitution. Insuch cases, polypeptides having such modifications exhibit increasedstability or a longer half-life relative to a polypeptide lacking suchan amino acid substitution.

In one embodiment, methods as disclosed herein may be represented asuses of the compositions as described herein for various therapeutic andprophylactic purposes as described herein, or alternatively, uses of thecompositions as described herein in the preparation of a medicament or atherapeutic composition or a composition for various therapeutic andprophylactic purposes as described herein.

In one embodiment, the compositions and methods as disclosed hereincomprise the various components or steps. However, in anotherembodiment, the compositions and methods as disclosed herein consistessentially of the various components or steps, where other componentsor steps may be included. In another embodiment, the compositions andmethods as disclosed herein consist of the various components or steps.

In some embodiments, the term “comprise” may encompass the inclusion ofother components of the composition which affect the efficacy of thecomposition that may be known in the art. In some embodiments, the term“consisting essentially of” comprises a composition, which has chimericantigen receptor-expressing T-cells (CAR T-cells), and apoptotic cellsor any apoptotic cell supernatant. However, other components may beincluded that are not involved directly in the utility of thecomposition. In some embodiments, the term “consisting” encompasses acomposition having chimeric antigen receptor-expressing T-cells (CART-cells), and apoptotic cells or an apoptotic cell supernatant asdisclosed herein, in any form or embodiment as described herein.

In one embodiment, “treating” comprises therapeutic treatment and“preventing” comprises prophylactic or preventative measures, whereinthe object is to prevent or lessen the targeted pathologic condition ordisorder as described hereinabove. Thus, in one embodiment, treating mayinclude directly affecting or curing, suppressing, inhibiting,preventing, reducing the severity of, delaying the onset of, reducingsymptoms associated with the disease, disorder or condition, or acombination thereof. Thus, in one embodiment, “treating,”“ameliorating,” and “alleviating” refer inter alia to delayingprogression, expediting remission, inducing remission, augmentingremission, speeding recovery, increasing efficacy of or decreasingresistance to alternative therapeutics, or a combination thereof. In oneembodiment, “preventing” refers, inter alia, to delaying the onset ofsymptoms, preventing relapse to a disease, decreasing the number orfrequency of relapse episodes, increasing latency between symptomaticepisodes, or a combination thereof. In one embodiment, “suppressing” or“inhibiting”, refers inter alia to reducing the severity of symptoms,reducing the severity of an acute episode, reducing the number ofsymptoms, reducing the incidence of disease-related symptoms, reducingthe latency of symptoms, ameliorating symptoms, reducing secondarysymptoms, reducing secondary infections, prolonging patient survival, ora combination thereof.

In one embodiment, a composition as disclosed herein is administeredonce. In another embodiment, the composition is administered twice. Inanother embodiment, the composition is administered three times. Inanother embodiment, the composition is administered four times. Inanother embodiment, the composition is administered at least four times.In another embodiment, the composition is administered more than fourtimes.

In one embodiment, CAR T-cells as disclosed herein are administeredonce. In another embodiment, CAR T-cells are administered twice. Inanother embodiment, CAR T-cells are administered three times. In anotherembodiment, CAR T-cells are administered four times. In anotherembodiment, CAR T-cells are administered at least four times. In anotherembodiment, the composition is administered more than four times.

In one embodiment, the composition as disclosed herein is a therapeuticcomposition. In another embodiment, the composition as disclosed hereinhas therapeutic efficacy.

In one embodiment, disclosed herein are a composition which providesreduced inflammatory cytokine or chemokine release compared to acomposition comprising CAR T-cells alone, but with comparablecytotoxicity compared to a composition comprising CAR T-cells alone.

A skilled artisan would appreciate that the term “about”, may encompassa deviance of between 0.0001-5% from the indicated number or range ofnumbers. Further, it may encompass a deviance of between 1-10% from theindicated number or range of numbers. In addition, it may encompass adeviance of up to 25% from the indicated number or range of numbers.

A skilled artisan would appreciate that the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “an agent” or “at least an agent” mayinclude a plurality of agents, including mixtures thereof.

Throughout this application, various embodiments disclosed herein may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicated number and asecond indicated number and “ranging/ranges from” a first indicatednumber “to” a second indicated number are used herein interchangeablyand are meant to include the first and second indicated numbers and allthe fractional and integral numerals there between.

The following examples are presented in order to more fully illustrateembodiments disclosed herein. They should in no way be construed,however, as limiting the broad scope of the disclosure.

EXAMPLES Example 1: Apoptotic Cell Therapy Prevents Cytokine Storms inSubjects Administered Car T-Cell Therapy

Materials and Methods

Recombinant DNA Constructs

A CAR is developed that retargets T-cell specificity against a specifictumor associated antigen. A control CAR is developed that directsT-cells to a non-related tumor associated antigen. The CARs used for thebackground are armored CAR T or 4^(th) generation CAR T-cells. In oneembodiment, cells are also engineered to express the ectodomain of theIL-4 receptor alpha subunit joined to the transmembrane and endodomainof the beta-chain used by the IL-2 and IL-15 receptors, allowing theT-cells to be expanded by addition of IL-4.

Retroviral Transduction and Culture of T4+ T-Cells

Blood samples are obtained from healthy volunteers and cancer patients.T-cells may be activated prior to gene transfer using CD3/CD28-coatedparamagnetic beads (1:1 bead/cell ratio; Life Technologies) or PHA (5mg/ml; Sigma-Aldrich). Retroviral transduction of activated T-cells isperformed using PG13 retroviral packaging cells. Where indicated,transduction is conducted using SFG T4 viral vector manufactured undergood manufacturing practice (GMP), followed by expansion of T-cellsusing GMP-grade IL-4 (30 ng/ml) in gas-permeable bags.

Cells and Cell Culture

A firefly luciferase-expressing tumor cell line is propagated inappropriate medium, for example, DMEM (Lonza, Basel, Switzerland)supplemented with 10% FBS (Sigma-Aldrich), GlutaMAX, andantibioticantimycotic solution (Life Technologies).

Flow Cytometry

Expression of CARs is detected using biotinylated Ab and streptavidin-PE(Life Technologies). To quantify tumor antigen expression in organs frommice, dissected tissues are homogenized in PBS using a syringe plungerand filtered through a 100-mm cell strainer. After treatment with redblood lysis solution (Miltenyi Biotec, Bisley, U.K.), cells are fixedusing 4% paraformaldehyde (37° C. for 10 min), permeabilized usingice-cold methanol for 30 min, and washed with 40% D10/60% PBS. Next,cells are incubated with rabbit anti-tumor antigen antibody or rabbitserum as control (Dako, Ely, U.K.) followed by swine F(ab9)2 anti-rabbitIgG-FITC (Dako). Alternatively, expression of human tumor antigenreceptors is demonstrated by flow cytometry. In all cases, forwardscatter/side scatter gates are used to identify the dominanT-cellpopulation present. Flow cytometry is performed using a FACSCalibur flowcytometer with CellQuest Pro software.

Cytokine Analysis

Supernatants and sera are analyzed using ELISA kits, cytometric beadarrays (Th1/Th2/Th17; BD Biosciences) as described by the manufacturers.For example, analysis may be fore pro-inflammatory cytokine, which inone case would be IL-6.

Cytotoxicity Assays

Destruction of tumor cell monolayers by T-cells is visualized by crystalviolet staining Tumor cell viability is quantified using an MTT assay(Life Technologies), as described by the manufacturer.

In Vitro Luciferase Assay

A total of 0.5×10⁶ transduced (and matched untransduced cells ascontrol) are assayed using a luciferase assay system kit (Promega,Madison, Wis.), as described by the manufacturer. Assays are read usinga microplate reader with Omega software.

In Vivo Studies

Tumor cells (1×10⁶) are inoculated into mice either i.p. in PBS or s.c.in 200 ml matrigel (BD Biosciences). Tumor engraftment is confirmed bybioluminescence imaging (BLI) and mice are sorted into groups withsimilar signal intensity prior to T-cell administration. Imaging isperformed using an IVIS Lumina II or Spectrum (PerkinElmer) with LivingImage software (PerkinElmer), using large binning for T-cell imaging. Toassess in vivo toxicity of T-cells, organs are collected from mice,formalin fixed, and subjected to histopathologic analysis.

Results

CAR T-Cells are Directed to and Destroy Tumor Cells Expressing theTarget of Interest

The tumor associated antigen of interest is expressed in healthy mousetissue at low levels and on the tumor expressed in the mouse.

In vitro, the human CAR T rapidly destroys tumor cell monolayers whilecontrol T-cells did not destroy the tumor cells. Additionally, cytokineproduction is observed following stimulation of the T-cells with thetumor cells expressing the tumor associate antigen. As a control,non-transformed mouse cells were tested for activation of human CART-cells. Non-transformed mouse cells also stimulate human CAR T-cells,demonstrating that they also express the tumor associated antigen.Non-transformed mouse cells do not stimulate human CAR T-cells.

CAR T-Cell Therapy Induces Cytokine Release Syndrome

Three groups of tumor-free mice as well as mice with tumors areadministered (i.p. or directly into the tumor) increasing doses of CART-cells (3×10⁶, 10×10⁶ or 30×10⁶). At the highest dose, tumor-free miceand mice with tumors demonstrate subdued behavior, piloerection, andreduced mobility within 24 h, accompanied by rapid weight loss followedby death within 48 hrs. Human interferon-gamma and mouse IL-6 aredetectable in blood samples from the mice given the highest dose of CART-cells. Animals that receive a high dose of CAR T-cells directed to adifferent tumor antigen do not exhibit weight loss or behavioralalterations.

Administration of Apoptotic Cells Inhibits or Reduces the Incidence ofCytokine Release Syndrome Induced by CAR T-Cell Therapy

One group of mice given the highest dose of CAR T-cells is concomitantlyadministered 2.10×10⁸/kg apoptotic cells, which was previouslydemonstrated to be a safe and effective dose. Mice receiving human CART+ apoptotic cells have significantly lowered levels of mouse IL-6,lower weight loss, and reduced mortality.

Example 2: Effect of Apoptotic Cells on Cytokine Storm without aNegative Effect on the CAR-T Cell Efficacy

Objective:

Test the effect of apoptotic cells or supernatants derived fromapoptotic cells on cytokine storm marker cytokines and CAR T-cellefficacy on tumor or cancer cells.

Methods:

A solid tumor model (van der Stegen et al., 2013 ibid) reported toinduce cytokine storms in mice was utilized. In this model, T cells wereengineered with a chimeric antigen receptor (CAR) targeting certain ErbBdimers (T4⁺ CAR-T cells), which are often highly up-regulated inspecific solid tumors such as head and neck tumors and ovarian cancers.T-cells were isolated from PBMC separated from peripheral blood usingCD3 micro-beads. Vectors containing the chimeric T4+ receptor wereconstructed and transducer into the isolated T-cells, resulting in T4+CAR T-cells. For the experiments performed herein, T4+ CAR T-cells werepurchased (Creative Biolabs (NY USA) or Promab Biotechnologies (CAUSA)). FIG. 3 presents verification of cell surface expression of 4αβ (achimeric cytokine receptor) on the T4+ CAR T-cells using flow cytometryand an anti-CD124 monoclonal antibody (Wilkie et al., ibid). Inaddition, a PCR procedure was performed and verified the presence of thevector in transduced T cells.

SKOV3-luc ovarian adenocarcinoma tissue culture cells were used (Wilkieet al., ibid). SKOV3-luc highly express ErbB receptors and are a targetfor the T4⁺ CAR-T cells (van der Stegen et al., 2013, ibid). TheseSKOV3-luc cells were manipulated to constitutively express the fireflyluciferase gene, allowing tracking of cell proliferation in vitro andtumor growth and recession in vivo.

Apoptotic Cells

An enriched mononuclear cell fraction was collected via a leukapheresisprocedure from a healthy, eligible donor. The procedure was performed atHadassah Ein Kerem apheresis unit. Collected cells were processed andstored in liquid nitrogen immediately following leukapheresiscompletion. For preparation of apoptotic cells (ApoCell), cryopreservedcells were thawed, washed, and then incubated at 37° C. in 5% CO₂ in thepresence of 50 mg/mL methyl prednisolone (Pfizer, NY, USA) and 10% ofautologous plasma for 6 hours. At the end of incubation, cells werecollected, washed and resuspended with the buffer of choice according todownstream applications. Apoptosis and viability of ApoCell weredetermined using AnnexinV and PI (MBL, MA, USA) staining (≥40% and ≤15%,respectively) via Flow cytometer. Results analyzed using FCS expresssoftware.

Apoptotic Cell Supernatants

Eight (8) million apoptotic cells per seeded per well in a 12-wellplate. After 24 hours the cells were centrifuge (290 g, 4 degreesCelsius, 10 minutes). Supernatant was collected and frozen in aliquotsat −80 degrees until use. Different numbers of cells are used to makesupernatants. Some aliquots contain concentrated supernatants.

Monocyte Isolation

PBMCs were isolated using Ficoll (GE healthcare, United Kingdome) fromperipheral blood\ buffy coat obtained from healthy, eligible donors.Cells were brought to a concentration of 15×10⁶ cells\ml in RPMI1640(Gibco, Thermo Fisher Scientific, MA, USA) and seeded in a 0.9 ml dropin the middle of 35 mm plates (Corning, N.Y., USA). Plates were thenincubated at 37° C. in 5% CO₂ for 1 hour. At the end of incubation,cells were washed three times with PBS (Biological industries, BeitHaemek, Israel) and adhesion was determined using a light microscope.Cells were then incubated with complete media (RPMI1640+10% heatinactivated FBS+1% Glutamax+1% PenStrep, all from Gibco).

An alternative method of monocyte isolation was also used wherein humanmononuclear cells were isolated from heparinized peripheral blood bydensity gradient centrifugation. The isolated mononuclear cells thenwere separated into monocyte, B-cell and T-cell populations bypositively selecting monocytes as the CD14+ fraction by magnetic beadseparation (Miltenyi Biotec., Auburn, Calif., USA), positively selectingB-cells as the CD22+ fraction, and negatively selecting T-cells as theCD14-CD22-fraction. Purity was greater than 95 percent for monocytes.

For macrophage differentiation, at the end of adhesion, cells werewashed three times with PBS then incubated with RPMI1640+1% Glutamax+1%PenStrep and 10% heat inactivated human AB serum (Sigma, MO, USA). Cellswere incubated at 37° C. and 5% for 7-9 days, with media exchange at day3 and day 6. Differentiation was determined by morphology via lightmicroscope.

Supernatant from Apo+ Monocytes

CD14+ monocytes were cultured with apoptotic cells as prepared above ata ratio of 1:16, for 24 h. The number of monocytes was: 0.5 millioncells per well in a 12-well plate and the number of apoptotic cells was:8 million cells per well in a 12-well plate. After incubation for 24hours the cells were centrifuge (290 g, 4 degrees Celsius, 10 minutes).Supernatant was collected and frozen in aliquots at −80 degrees untiluse. Similar procedures could be performed at different ratios ofmonocytes:apoptotic cells and/or using other sources of cells, such asmacrophages and dendritic cells.

Results:

Step 1: Verification of T4⁺ CAR-T Cell Activity Against SKOV3-Luc TumorCells

To corroborate the T4⁺ CAR-T cell activity, monolayers of SKOV3-luc wereexposed to either 1,000,000 (one million) T4⁺ CAR-T cells or to1,000,000 (one million) non-transduced T cells. After 24 h incubation,T4⁺ CAR-T cells reduced SKOV3-luc proliferation by 30% compared to thenon-transduced T cell control (FIG. 4), showing anti-tumor activity ofthe T4⁺ CAR-T cells.

Step 2: Activity of Stand-Alone T4+ CAR-T Cells Against SKOV3-Luc TumorCells was Compared to Activity Post Exposure to Apoptotic Cells

Apoptotic cells (ApoCell) and apoptotic cell supernatants (ApoSup andApoMon Sup) were tested to determine if they interfere with T4+ CAR-Tcell anti-tumor activity. The SKOV3-luc tumor cells were incubate withApoptotic Cells for one hour, followed by the addition of T4+ CAR-Tcells (500,000, five hundred thousands) or T4+ non-transduced T cells(500,000, five hundred thousands) (ratio of 1:2 T4⁺ CAR-T cells toApoptotic Cells). The tumor cell/Apoptotic cell/T4⁺ CAR T-cells werethen co-cultured for 48 h. The control SKOV3-luc tumor cells wereco-cultured with T4+ CAR-T cells and Hartman solution (the vehicle ofApoptotic Cells), but without Apoptotic Cells, for 48 h.

The results showed that after 48 h incubation, T4+ CAR-T cellsanti-tumor activity was superior to incubation with non-transduced Tcells. Similar incubations were performed with apoptotic cellsupernatants. Surprisingly, T4+ CAR T-cell anti-tumor activity wascomparable with or without exposure to apoptotic cells or apoptotic cellsupernatants. (FIG. 5).

Step 3: Effect of Apoptotic Cells on Amelioration, Reduction orInhibition of Cytokine Storms Resulting from CAR-T Treatment

The effect of apoptotic cells to reduce cytokine storms was examinednext. IL-6 is a prototype pro-inflammatory cytokine that is released incytokine storms (Lee D W et al. (2014) Blood 124(2): 188-195) and isoften used as a marker of a cytokine storm response.

Cultures were established to mimic an in vivo CAR T-cell therapyenvironment. SKOV3-luc tumor cells were cultured in the presence ofhuman monocyte-macrophages and T4+ CAR T-cells. The concentration ofII-6 measured in the culture media was approximately 500-600 pg/ml. Thisconcentration of IL-6 is representative of a cytokine storm.

Unexpectedly, IL-6 levels measured in the cultured media of SKOV3-luctumor cells, human monocyte-macrophages, T4+ CAR-T cells, wherein thetumor cells had been previously incubated with apoptotic cells for onehour (ratio of 1:2 T4+ CAR-T cells to Apoptotic Cells) were dramaticallyreduced. Similarly, IL-6 levels measured in the cultured media ofSKOV3-luc tumor cells, human monocyte-macrophages, T4+ CAR-T cells,wherein the tumor cells had been previously incubated with apoptoticcell supernatants for one hour, were also dramatically reduced. Thisreduction in concentration of IL-6 is representative of a decrease inthe cytokine storm (FIG. 6).

It was concluded that unexpectedly, apoptotic cells and apoptoticsupernatants do not abrogate the effect of CAR-T cells on tumor cellproliferation while at the same time they down regulatingpro-inflammatory cytokines such as IL-6, which was been described as amajor cytokine leading to morbidity.

Step 4: Analysis Using a Wider Range of Cytokines

To further evaluate the effect on a possible wider range and levels ofcytokines that are not generated during experimental procedures but doappear in clinical settings during a human cytokine storm, LPS (10ng/ml) was added to the SKOV3-luc culture conditions outlined above. Theaddition of LPS is expected to exponentially increase the cytokine stormlevel. As expected, the addition of LPS increased the cytokine stormeffect and as a result IL-6 levels increased to approximately 30,000pg/ml. Other cytokines known to be expressed in high levels during acytokine storm showed elevated levels, for example: TNF-α (250-300pg/ml), IL-10 (200-300 pg/ml), IL1-alpha (40-50 pg/ml) and IL-18 (4-5pg/ml). As shown in FIG. 7, exposure to apoptotic cells dramaticallyreduced the levels of IL-6 even during the exponential state of thecytokine storm to almost normal levels that may be seen in clinicalsettings and is not always seen in experimental procedures with CART-cells. This effect was similar across the other pro-inflammatorycytokines with normalization of levels of cytokines TNF-alpha, IL-10,IL1-alpha, IL-1beta, and IL-18 with reduction between 20-90%. Similarresults were found when using apoptotic cell supernatants in place ofthe apoptotic cells.

CONCLUSION

CAR-T cell therapy has been documented to cause cytokine storms in asignificant number of patients. These results presented here demonstratethat surprisingly, apoptotic cells and apoptotic cell supernatants wereable to decrease cytokine storms without affecting CAR-T cell efficacyto kill tumor cells.

While certain features disclosed herein have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritdisclosed herein.

The invention claimed is:
 1. A method of inhibiting or reducing theincidence of a cytokine release syndrome (CRS) or a cytokine storm in asubject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) cancer therapy, the method comprising the step of administeringa composition to said subject, the composition comprising (a) chimericantigen receptor-expressing T-cells (CAR T-cells) and a pharmaceuticallyacceptable excipient, and (b) either early apoptotic cells or an earlyapoptotic cell supernatant, and a pharmaceutically acceptable excipient,wherein said early apoptotic cells are ≥40% AnnexinV⁺ and ≤15% propidiumiodide⁺, and wherein said early apoptotic cells are peripheral bloodmononuclear cells.
 2. The method of claim 1, wherein said earlyapoptotic cells are autologous to the subject or are pooled third-partydonor cells.
 3. The method of claim 1, wherein said method furthercomprises administering an additional agent selected from the groupcomprising a CTLA-4 blocking agent, a tellurium-based compound, analpha-1 anti-trypsin or fragment thereof or analogue thereof, or animmune modulating agent, or any combination thereof.
 4. The method ofclaim 3, wherein administration of said additional agent occurs prior tothe CAR T-cell therapy.
 5. The method of claim 3, wherein administrationof said additional agent occurs concurrent with the CAR T-cell therapy.6. The method of claim 3, wherein administration of said additionalagent occurs following the CAR T-cell therapy.